Human poly (ADP-ribose) polymerase 2 materials and methods

ABSTRACT

The invention provides a novel human poly(ADP-ribose) polymerase (hPARP2) polypeptides, polynucleotides encoding the polypeptides, expression constructs comprising the polynucleotides, and host cells transformed with the expression constructs. Also provided are methods for producing the hPARP2 polypeptides, antibodies that are immunoreactive with the hPARP2 polypeptides. In addition, there are provided methods for identifying specific binding partners of hPARP2, and more particularly methods for identifying binding partners that modulate biological activity of hPARP2. Methods of modulating biological activity of hPARP2 in vitro and in vivo are also provided.

[0001] This application claims the benefit of U.S. ProvisionalApplication Serial No. 60/139,543, filed Jun. 16, 1999.

[0002] The present invention relates generally to a novel humanpolypeptide having poly(ADP-ribose) polymerase activity, topolynucleotides encoding the polypeptide, and to methods of using suchmaterials.

BACKGROUND OF THE INVENTION

[0003] Regulation of gene function occurs by several mechanisms ineukaryotic cells. Amongst these mechanisms are gene transcriptionregulation, mRNA translation regulation, and post-translationmodification of proteins. Post-translation modification of proteinsincludes several processes whereby proteins are covalently altered toaffect cellular, sub-cellular localization, stability, transport,interaction specificity, enzymatic activity, and numerous othercharacteristics.

[0004] Common and extensively studied covalent modification processesinclude acetylation, glycosylation, and phosphorylation. Less wellcharacterized is a process that involves the covalent addition ofpolymers of ADP-ribose to protein targets. The polymer is termed“poly(ADP-ribose),” and the enzyme(s) responsible for this activity havebeen variously called poly(ADP-ribose) polymerase (PARP),poly(ADP-ribose) synthetase (PARS), or ADP-ribosyl transferase (ADPRT)[Althaus and Richter, ADP-Ribosylation of Proteins: Enzymology andBiochemical Significance, Molecular Biochemistry and Biophysics,Springer-Verlag (1987)]. A previously identified PARP gene product(hereinafter “PARP1”) is expressed at high levels in the nuclei of cellsand is dependent upon DNA damage for activation [Szab6 and Dawson,Trends Pharmacol Sci 19(7):287-98 (1998)]. Current models hypothesizethat PARP1 binds to DNA single or double stranded breaks through anamino terminal DNA binding domain. The binding activates the carboxyterminal catalytic domain and results in the formation of polymers ofADP-ribose on target molecules. PARP1 is itself a target ofpolyADP-ribosylation by virtue of a centrally located automodificationdomain. The ribosylation of PARP1 causes dissociation of the PARP1molecules from the DNA. The entire process of binding, ribosylation, anddissociation occurs very rapidly. It has been suggested that thistransient binding of PARP1 to sites of DNA damage may result in therecruitment of DNA repair machinery or may act to suppress recombinationlong enough for the recruitment of repair machinery [Satoh and Lindahl,Nature 356(6367):356-8 (1992)].

[0005] The source of ADP-ribose for the PARP reaction is nicotinamideadenine dinucleotide (NAD). NAD is synthesized in cells from cellularATP stores and thus high levels of activation of PARP activity canrapidly lead to depletion of cellular energy stores. It has beendemonstrated that induction of PARP activity can lead to cell death thatis correlated with depletion of cellular NAD and ATP pools [Yamamoto etal., Nature 294(5838):284-6 (1981); Sims et al., Biochemistry22(22):5188-94 (1983)]. PARP activity is induced in many instances ofoxidative stress or during inflammation. For example, during reperfusionof ischemic tissues reactive nitric oxide is generated and nitric oxideresults in the generation of additional reactive oxygen speciesincluding hydrogen peroxide, peroxynitrate and hydroxyl radical [Szabó,Eur J Pharmacol 350(1):1-19 (1998)]. These latter species can directlydamage DNA and the resulting damage induces activation of PARP activity.Frequently, it appears that sufficient activation of PARP activityoccurs so that the cell energy stores are depleted and the cell dies.

[0006] A similar mechanism is believed to operate during inflammationwhen endothelial cells and pro-inflammatory cells synthesize nitricoxide which results in oxidative DNA damage in surrounding cells and thesubsequent activation of PARP activity [Szabó 1998, supra]. Such amechanism is also believed to play a role in tissue damage associatedwith transient cerebral ischemia, in which excessive NMDA receptoractivation mediates DNA damage, which induces excessive PARP activation[Lo et al., Stroke 29:830-6 (1998)]. The cell death that results fromPARP activation is believed to be a major contributing factor in theextent of tissue damage that results from ischemia/reperfusion injury orfrom inflammation.

[0007] Two lines of evidence suggest that PARP activity is a criticalelement in those processes. First, chemical inhibitors of PARP activityhave been successfully used to reduce tissue damage resulting in animalmodels of ischemia/reperfusion injury or inflammation. Second, mice inwhich both alleles of PARP1 have been disabled (PARP1 knockout mice) areresistant to numerous forms of ischemia/reperfusion injury anddetrimental effects of inflammation. Because of those observations,potent small molecule inhibitors of PARP activity have great potentialas clinical drug candidates in several indications.

[0008] The experimental data derived from the PARP1 knockout micesuggest that inhibitors of PARP1 function may be clinically beneficial.However, a recent report demonstrates that PARP1 knockout mice are notdevoid of DNA damage-inducible PARP activity [Shieh et al., J Biol Chem273(46):30069-72 (1998)]. A recently identified gene product, tankyrase,has been shown to have poly(ADP) ribosylation activity [Smith et al.,Science 282(5393):1484-7 (1998)]. Tankyrase activity however, does notappear to be inducible by DNA damage and thus is unlikely to account forthe activity observed in PARP1 knockout mice. It has been suggested thatthe residual DNA damage induced PARP activity in PARP1 knockout mice maybe due to the activity of a second PARP gene, which has been identifiedin the mouse and named murine PARP2 [Shieh et al. (1998), supra]. Theexistence of multiple PARP genes in mammals suggests that appropriatedrug design for human therapeutics requires the identification ofadditional human gene products with PARP activity. A gene comparable tomouse PARP2 has not previously been identified in humans.

[0009] In view of the above considerations, it is clear that existingknowledge is lacking with respect to cellular DNA repair mechanisms,signaling and induction of cell death in response to DNA damage,mechanisms of inflammation, and treatment of inflammation-mediateddisease states. Thus, there exists a need in the art for theidentification of additional human PARP-like molecules for use indetermining the selectivity of therapeutics designed to inhibit PARPfunction and as targets in their own right for therapeutic interventionin human diseases. The profiling of PARP inhibitors on additional PARPgene products may allow for the PARP-selective drugs, which could bebeneficial for particular indications, the reduction of undesirable sideeffects, or the targeting of therapeutics to selected tissues. Likewise,the identification of hPARP2 will allow for the development of hPARP2specific therapeutics, which may also have benefits in terms ofparticular disease indications, the reduction of undesirable sideeffects, and the targeting of therapeutics to particular tissues. Theidentification of human hPARP2 would also allow for the development ofdrugs with the ability to inhibit both PARP activities that may alsohave therapeutic benefit. Other purposes and advantages of the inventionwill be readily apparent to the artisan having ordinary skill in theart.

SUMMARY OF THE INVENTION

[0010] It has now been discovered that these and other purposes can beachieved by the present invention, which, in one aspect, is a purifiedand isolated hPARP2 polypeptide comprising an amino acid sequencedefined in SEQ ID NO:2 or a functional derivative thereof The inventionalso embraces hPARP2 polypeptides encoded by a polynucleotide whichhybridizes under stringent (moderately or highly) conditions to thecomplement of the polynucleotide set out in SEQ ID NO:2 and apolynucleotide which hybridizes under stringent (moderately or highly)conditions to the complement of a polynucleotide that encodes thepolypeptide set out in SEQ ID NO:1.

[0011] In another aspect, the invention further provides polynucleotidesencoding the hPARP2 polypeptide defined in SEQ ID NO:2. Preferably, thepolynucleotides comprise the nucleotide sequence defined in SEQ ID NO:1.Alternatively, the polynucleotides encoding an hPARP2 polypeptide can bepolynucleotides that hybridize under moderately stringent hybridizationconditions to the coding or non-coding strand of the polynucleotide ofSEQ ID NO:1 or a polynucleotide which hybridizes to the complement of apolynucleotide that encodes the polypeptide set out in SEQ ID NO:2. In apreferred case, the polynucleotide hybridizes to the complement of thepolynucleotide defined in SEQ ID NO:1, under stringent (moderately orhighly) hybridization conditions, and encodes a protein that haspoly(ADP) polymerase activity or interacts with damaged DNA. Thepolynucleotides of the invention may be DNA molecules or RNA molecules,and may optionally further comprise a detectable label moiety.

[0012] In another aspect, the invention provides expression constructsthat comprise an hPARP2-encoding polynucleotide having a nucleotidesequence defined in SEQ ID NO:1 or a polynucleotide which hybridizes tothe complement of a polynucleotide that encodes the polypeptide set outin SEQ ID NO:2. The hPARP2-encoding polynucleotide can be operativelylinked to a heterologous promoter. Host cells transformed or transfectedwith an hparp2-based expression construct are also provided. Alsocontemplated are methods for producing a polypeptide having an aminoacid sequence defined by SEQ ID NO:2, comprising the steps of:

[0013] a) growing hPARP2-expressing host cells under conditionsappropriate for expression of the polypeptide; and

[0014] b) isolating the polypeptide from the host cell or the medium inwhich the host cell is grown.

[0015] The invention further provides antibodies that are specificallyimmunoreactive with the hPARP2 polypeptides described herein. As usedherein, “specifically immunoreactive” antibodies embrace those whichonly recognize the polypeptide (or antibody, as discussed below) of theinvention. The antibody can be selected from the group consisting ofmonoclonal antibodies, polyclonal antibodies, single chain antibodies(scFv antibodies), chimeric antibodies, multifunctional/multispecificantibodies, humanized antibodies, human antibodies, CDR-graftedantibodies, Fab fragments, Fab′ fragments, F(ab′)₂ fragments, Fvfragments, diabodies; linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibody fragments.Also provided are cell lines that produce antibodies. Anti-idiotypeantibodies specifically immunoreactive with an hPARP2-specific antibodyare also contemplated.

[0016] In a further aspect, the invention is a method for identifying aspecific binding partner of an hPARP2 polypeptide, comprising:

[0017] a) contacting the hPARP2 polypeptide with a test compound underconditions that permit binding of the hPARP2 polypeptide and the testcompound;

[0018] b) detecting binding of the test compound and the hPARP2polypeptide; and

[0019] c) identifying the test compound as a specific binding partner ofthe hPARP2 polypeptide.

[0020] The specific binding partner is preferably a compound thatmodulates a biological activity of the hPARP2 polypeptide. Accordingly,the specific binding partner can be one that inhibits a biologicalactivity of the hPARP2 polypeptide. Alternatively, the specific bindingpartner can be one that enhances a biological activity of the hPARP2polypeptide.

[0021] In still another aspect, the invention is a method foridentifying a specific binding partner of an hparp2 polynucleotide,comprising:

[0022] a) contacting the hparp2 polynucleotide with a test compoundunder conditions that permit binding of the hparp2 polynucleotide andthe test compound;

[0023] b) detecting binding of the test compound and the hparp2polynucleotide; and

[0024] c) identifying the test compound as a specific binding partner ofthe hparp2 polynucleotide.

[0025] Thus, specific binding partner identified by the method ispreferably a compound that modulates expression of the hPARP2polypeptide. The specific binding partner can be one that inhibitsexpression of the hPARP2 polypeptide or one that enhances expression ofthe hPARP2 polypeptide.

[0026] Furthermore, in another aspect, the invention is a method oftreating a human subject having a medical condition mediated by apoly(ADP-ribose) polymerase, comprising administering to the subject anhPARP2 inhibitory compound in an amount effective for inhibiting hPARP2in the subject. The method may further comprise administering to thesubject an hPARP1 inhibitory compound in an amount effective forinhibiting hPARP1 in the subject.

[0027] In another aspect, the invention is a method of treating a humansubject having a disorder selected from the group consisting ofinflammatory disorders, neurological disorders, cardiovasculardisorders, and disorders of neoplastic tissue growth. Preferably thedisorder is an inflammatory disorder or is associated with inflammatorycell activation. For example, the medical condition can be a conditionthat is characterized by reperfusion injury. Illustrative disorderscharacterized by reperfusion injury include ischemic stroke, hemorrhagicshock, myocardial ischemia or infarction, transplantation, and cerebralvasospasm. Other disorders associated with inflammatory cell activationand amenable to treatment according to the methods of the inventioninclude rheumatoid arthritis, osteoarthritis, gouty arthritis,spondylitis; Behcet disease; sepsis, septic shock, endotoxic shock, gramnegative sepsis, gram positive sepsis, toxic shock syndrome; multipleorgan injury syndrome secondary to septicemia, trauma, or hemorrhage;allergic conjunctivitis, vernal conjunctivitis, uveitis,thyroid-associated ophthalmopathy; eosinophilic granuloma; asthma,chronic bronchitis, allergic, ARDS, chronic obstructive pulmonarydisease, silicosis, pulmonary sarcoidosis, pleurisy, alveolitis,vasculitis, pneumonia, bronchiectasis, pulmonary oxygen toxicity;reperfusion injury of the myocardium, brain, or extremities; cysticfibrosis; keloid formation, scar tissue formation; atherosclerosis;systemic lupus erythematosus, autoimmune thyroiditis, multiplesclerosis; Reynaud's syndrome; graft versus host disease, allograftrejection; chronic glomerulonephritis; inflammatory bowel disease,Crohn's disease, ulcerative colitis, necrotizing enterocolitis;inflammatory dermatoses, contact dermatitis, atopic dermatitis,psoriasis, urticaria, fever and myalgias due to infection; meningitis,encephalitis, and brain or spinal cord injury due to minor trauma;Sjögren's syndrome; diseases involving leukocyte diapedesis; alcoholichepatitis; bacterial pneumonia; antigen-antibody complex mediateddiseases; hypovolemic shock; Type I diabetes mellitus; acute and delayedhypersensitivity; disease states due to leukocyte dyscrasia andmetastasis; thermal injury; granulocyte transfusion associatedsyndromes; and cytokine-induced toxicity.

[0028] The invention, in still another aspect, is a method of inhibitingpoly(ADP-ribose) polymerase activity in a cell, comprising contactingsaid cell with an hPARP2 antagonist in an amount effective forinhibiting expression or activity of the hPARP2 polypeptide.

[0029] These and other features and advantages of the present inventionwill be appreciated from the detailed description and examples that areset forth herein. The detailed description and examples are provided toenhance the understanding of the invention, but are not intended tolimit the scope of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The present invention relates generally to a previouslyuncharacterized nucleic acid encoding a novel human protein designated“human poly(ADP-ribose) polymerase 2” (hereinafter “hPARP2”). Asillustrated herein hPARP2 is distinct from known proteins sharing apoly(ADP-ribose) polymerase activity. The present invention is based onthe discovery of novel gene that encodes the hPARP2 protein, and nucleicacid sequences, oligonucleotides, fragments, and antisense moleculesthereof.

[0031] The nucleotide sequence information provided by the inventionmakes possible large-scale expression of the encoded hPARP2 polypeptideby techniques well known and routinely practiced in the art. Theinvention also permits identification and isolation of polynucleotidesencoding related hPARP2 polypeptides by well-known techniques includingSouthern (DNA) and/or northern (mRNA) hybridization, and amplificationtechniques such as polymerase chain reaction (PCR), ligase chainreaction (LCR), and the like. Examples of related polynucleotidesinclude human and non-human parp2 genomic sequences, including allelicvariants, as well as polynucleotides encoding polypeptides homologous tohPARP2 and structurally related polypeptides sharing one or morebiological, immunological, and/or physical properties of hPARP2.

[0032] The invention includes both naturally-occurring and non-naturallyoccurring hPARP2 polynucleotides and polypeptide products thereof.Naturally-occurring hPARP2 products include distinct polynucleotide andpolypeptide hPARP2 species as they occur in humans. However, theinvention includes other human hPARP2 species defined through theanalysis of sequence homology. The invention further comprisescorresponding homologs of human hPARP2 that are expressed in cells ofother animal species, preferably mammalian homologs, and more preferablyprimate homologs. Within each hPARP2 species, the invention furtherprovides splice variants, which are encoded by the same genomic DNA butarise from distinct mRNA transcripts. Non-naturally occurring hPARP2products include variants of the naturally occurring hPARP2 productssuch as polynucleotide and polypeptide analogs (i.e., wherein one ormore nucleotides or amino acids are added, substituted, or deleted).Non-naturally-occurring hPARP2 products also include hPARP2 productsthat have been covalently modified, e.g., water-soluble polymermodifications, glycosylation variants, and the like.

[0033] The hPARP polypeptides and the nucleic acids that encode thepolypeptides provide a basis for diagnostic methods for the precise andaccurate detection and/or quantitation of hPARP2 expression and medicalconditions associated with excessive or insufficient hPARP2 activity.Furthermore, the nucleotide sequences disclosed herein may be used inthe detection of aberrations, such as mutations and deletions, in thegene encoding hPARP2. For example, the nucleotide sequences disclosedherein may be used to identify and isolate a genomic sequence forhparp2. PCR primers can be designed from various portions of the intronsand exons of a genomic hparp2 nucleic acid sequence that will allowdetection of aberrations in the genomic sequence.

[0034] The invention further provides methods of using hPARP2 andgenetically engineered host cells that express recombinant hPARP2 toevaluate and screen for modulators of the poly(ADP-ribose) polymeraseactivity of the enzyme. Such screening methods may be used for theidentification of allosteric agonists and antagonists of hPARP2 activityas well as for the identification of direct (e.g., competitiveinhibitors) of such activity. hPARP2 protein antagonists and inhibitors,such as anti-hPARP2 antibodies and hparp2 antisense molecules, willprovide the basis for pharmaceutical compositions for the treatment andamelioration of symptoms associated with excessive poly(ADP-ribose)polymerase activity. Agonists of hPARP2 will provide the basis of thetreatment and amelioration of symptoms associated with insufficientpoly(ADP-ribose) polymerase activity.

[0035] hparp2 Polynucleotides

[0036] The present invention provides, inter alia, novel purified andisolated polynucleotides encoding human hPARP2 polypeptides. Thepolynucleotides of the invention include DNA sequences and RNAtranscripts, both sense and complementary antisense strands, and splicevariants thereof. DNA sequences of the invention include, withoutlimitation, cDNA and genomic sequences. As used herein, lower case“hparp2” refers to a nucleic acid sequence whereas upper case “hPARP2”refers to an amino acid sequence.

[0037] “Nucleic acid” as used herein refers to an oligonucleotide orpolynucleotide sequence, and fragments or portions thereof, and to DNAor RNA of genomic or synthetic origin, which may be double-stranded orsingle-stranded, whether representing the sense or antisense strand. Anexemplary double-stranded polynucleotide according to the invention canhave a first strand (i.e., a coding strand) having a sequence encodingan hPARP2 polypeptide, along with a second strand (i.e., a“complementary” or “non-coding” strand) having a sequence deducible fromthe first strand according to the Watson-Crick base-pairing rules forDNA. Double-stranded or “duplex” structures may be DNA:DNA, DNA:RNA, orRNA:RNA nucleic acids. A preferred double-stranded polynucleotide is acDNA having a nucleotide sequence defined by SEQ ID NO:1. An exemplarysingle-stranded polynucleotide according to the invention is a messengerRNA (mRNA) encoding an hPARP2 polypeptide. Another exemplarysingle-stranded polynucleotide is an oligonucleotide probe or primerthat hybridizes to the coding or non-coding strand of a polynucleotidedefined by SEQ ID NO:1. Other alternative nucleic acid structures, e.g.,triplex structures, are also contemplated.

[0038] Genomic DNA of the invention comprises the protein-coding regionfor an hPARP2 polypeptide and includes allelic variants of the preferredpolynucleotides of the invention, such as single nucleotidepolymorphisms. Genomic DNA of the invention is distinguishable fromgenomic DNAs encoding polypeptides other than hPARP2 in that it includesthe hPARP2-coding region found in hparp2 cDNA of the invention. GenomicDNA can be transcribed into RNA, and the resulting RNA transcript mayundergo one or more splicing events wherein one or more introns (i.e.,non-coding regions) of the transcript are removed, or “spliced out.” RNAtranscripts that can be spliced by alternative mechanisms and thereforebe subjected to removal of different non-coding RNA sequences but stillencode an hPARP2 polypeptide, are referred to in the art as “splicevariants,” and are embraced by the invention. Splice variantscomprehended by the invention, therefore, are encoded by the same DNAsequences but give rise to different amino acid sequences. Such splicevariants can comprise regions in which the reading frame is shifted,wherein a downstream portion of the RNA sequence is translateddifferently, to yield different amino acid sequences in the resultingpolypeptides. Allelic variants are known in the art to be modified formsof the wild-type (predominant) gene sequence. Such modifications resultfrom recombination during chromosomal segregation or exposure toconditions that give rise to genetic mutation. Allelic variants, likewild-type genes, are naturally occurring sequences, as opposed tonon-naturally occurring variants, which arise from in vitromanipulation.

[0039] The invention also comprehends cDNA, which is obtained throughreverse transcription of an RNA polynucleotide encoding hPARP2 followedby second strand synthesis of a complementary strand to provide a doublestranded DNA. For example, the invention provides a cDNA sequence thatencodes a polypeptide having the amino acid sequence defined by SEQ IDNO:2. In a preferred embodiment, the invention provides polynucleotidescomprising a nucleotide sequence defined by SEQ ID NO:1.

[0040] In another aspect, the invention provides, inter alia,polynucleotides that encode polypeptides comprising amino acids 49-583,and preferably amino acids 1-583, of the amino acid sequence defined bySEQ ID NO:2. Alternatively, the invention provides polynucleotides thatencode polypeptides comprising amino acids 1-49 of the amino acidsequence defined SEQ ID NO:2. Exemplary polynucleotides relating to thisaspect of the invention include polynucleotides comprising at leastnucleotides 209-1811, and preferably nucleotides 63-1811, of thenucleotide sequence defined by SEQ ID NO:1. Alternative exemplarypolynucleotides comprise nucleotides 1-209 or 63-209 of the nucleotidesequence defined by SEQ ID NO:1. Preferably, the polynucleotides encodean hPARP2 polypeptide or fragments thereof having hPARP2 catalyticactivity.

[0041] As noted, highly preferred nucleic acid sequence according to theinvention is defined by SEQ ID NO:1. However, because the genetic codeis redundant or “degenerate” in its information-encoding properties,different nucleotide sequences may encode the same polypeptide sequence.Accordingly, the invention comprises the alternative (degenerate)nucleotide sequences that encode hPARP2 polypeptides of the inventionand functional equivalents thereof. For example, the invention includespolynucleotides comprising nucleotide sequences that are substantiallyhomologous to the hparp2 sequence of SEQ ID NO:1. More particularly, theinvention includes polynucleotides whose corresponding nucleotidesequences have at least 90%, preferably at least 95%, more preferably atleast 98%, and still more preferably at least 99% identity with anucleotide sequence defined in SEQ ID NO:1.

[0042] Variant polynucleotides of the invention further includefragments of the nucleotide sequence defined in SEQ ID NO:1 and homologsthereof. The disclosure of full-length polynucleotides encoding hPARP2polypeptides makes readily available to the person having ordinary skillin the art every possible fragment of the full-length polynucleotides.Preferably, fragment polynucleotides of the invention comprise sequencesunique to the hPARP2-coding nucleotide sequence, and therefore hybridizeunder highly stringent or moderately stringent conditions only (i.e.,specifically) to polynucleotides encoding hPARP2 or fragments thereofcontaining the unique sequence. Polynucleotide fragments of genomicsequences of the invention comprise not only sequences unique to thecoding region, but also include fragments of the full-length sequencederived from introns, regulatory regions, and/or other untranslatedsequences. Sequences unique to polynucleotides of the invention arerecognizable through sequence comparison to other known polynucleotides,and can be identified through use of computer software routinely used inthe art, e.g., alignment programs available in public sequencedatabases.

[0043] The invention also provides fragment polynucleotides that areconserved in one or more polynucleotides encoding members of the hPARP2family of polypeptides. Such fragments include sequences characteristicof the family of hPARP2 polypeptides, referred to as “signature”sequences. The conserved signature sequences are readily discernablefollowing simple sequence comparison of polynucleotides encoding membersof the hPARP2 family. Polynucleotide fragments of the invention can belabeled in a manner that permits their detection, including radioactiveand non-radioactive labeling.

[0044] Hybridization can be defined to include the process of formingpartially or completely double-stranded nucleic acid molecules throughsequence-specific association of complementary single-stranded nucleicmolecules. The invention, therefore, further encompasses the use ofnucleic acid species that hybridize to the coding or non-coding strandsof a polynucleotide that encodes an hPARP2 protein. Preferredhybridizing species hybridize to the coding or non-coding strand of thenucleotide sequence defined by SEQ ID NO:1. Also encompassed are speciesthat would hybridize to an hPARP2-encoding polynucleotide but for theredundancy of the genetic code, i.e., polynucleotides that encode thesame amino acid sequence but rely on different codon usuage.

[0045] Hybridizing species include, for example, nucleic acidhybridization or amplification probes (oligonucleotides) that arecapable of detecting nucleotide sequences (e.g., genomic sequences)encoding hPARP2 or closely related molecules, such as alleles. Thespecificity of the probe, i.e., whether it is derived from a highlyconserved, conserved, or non-conserved region or domain, and thestringency of the hybridization or amplification conditions (high,intermediate, or low) will determine whether the probe identifies onlynaturally occurring hparp2, or related sequences. Probes for thedetection of related nucleotide sequences are selected from conserved orhighly conserved regions of hparp2 family members and such probes may beused in a pool of degenerate probes. For the detection of identicalnucleotide sequences, or where maximum specificity is desired,oligonucleotide probes are selected from the non-conserved nucleotideregions or unique regions of hparp2 polynucleotides. As used herein, theterm “non-conserved nucleotide region” refers to a nucleotide regionthat is unique to hparp2 disclosed herein and does not occur in relatedhparp2 family members.

[0046] Specificity of hybridization is typically characterized in termsof the degree of stringency of the conditions under which thehybridization is performed. The degree of stringency of hybridizationconditions can refer to the melting temperature (T_(m)) of the nucleicacid binding complex [see, e.g., Berger and Kimmel, “Guide to MolecularCloning Techniques,” Methods in Enzymology, Vol. 152, Academic Press,San Diego Calif. (1987)]. “Maximal stringency” typically occurs at aboutT_(m) −5° C. (5° C. below the T_(m) of the probe); “high stringency” atabout 5° C. to 10° C. below T_(m); “intermediate stringency” at about10° C. to 20° C. below T_(m); and “low stringency” at about 20° C. to25° C. below T_(m).

[0047] Alternatively, the stringency of hybridization can refer to thephysicochemical conditions employed in the procedure. To illustrate,exemplary moderately stringent hybridization conditions are:hybridization in 3× saline sodium citrate (SSC), 0.1% sarkosyl, and 20mM sodium phosphate, pH 6.8, at 65° C.; and washing in 2×SSC with 0.1%sodium dodecyl sulfate (SDS), at 65° C. Exemplary highly stringenthybridization conditions are: hybridization in 50% formamide, 5×SSC, at42° C. overnight, and washing in 0.5×SSC and 0.1% SDS, at 50° C. It isunderstood in the temperature and buffer, or salt concentration [seeAusubel et al. (Eds.), Current Protocols in Molecular Biology, JohnWiley & Sons (1994), at pp. 6.0.3-6.4.10]. Modifications inhybridization conditions can be determined empirically or calculatedprecisely based on the length of the oligonucleotide probe and thepercentage of guanosine/cytosine (GC) base pairing of the probe. Thehybridization conditions can be calculated as described in Sambrook etal., (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press: Cold Spring Harbor, N.Y. (1989), at pp. 9.47-9.51.

[0048] The artisan will appreciate that hybridization under morestringent conditions enables the identification of species having ahigher degree of homology or sequence identity with the target sequence.By contrast, hybridization under less stringent conditions enablesidentification of species having a lesser but still significant degreeof homology or sequence identity with the target sequence. Therefore,also included within the scope of the present invention are nucleic acidspecies that are capable of hybridizing to the nucleotide sequence ofSEQ ID NO:1 under conditions of intermediate (moderate) to maximalstringency. Preferably, the hybridizing species hybridize to the codingor non-coding strands of a polynucleotide defined by SEQ ID NO:1 underhighly stringent conditions. Preferred polynucleotides include, interalia, species that hybridize to a nucleic acid sequence consisting ofnucleotides 1 to 209 as defined by SEQ ID NO:1 or the complement of thatregion under conditions of moderate stringency, and more preferably thatdo so under conditions of high stringency. Other preferredpolynucleotides are species that hybridize to a nucleic acid sequenceconsisting of nucleotides 63 to 209 as defined by SEQ ID NO:1 or thecomplement of that region under conditions of moderate stringency, andmore preferably that do so under conditions of high stringency.

[0049] The polynucleotides of the invention encompass oligonucleotides(i.e., nucleic acid oligomers typically about 10 to 60 nucleotides inlength) that hybridize to either the coding or the non-coding strands ofa nucleic acid encoding an hPARP2 amino acid sequence. In particular,the invention comprises oligonucleotides that hybridize to the coding ornon-coding strand of a polynucleotide defined by SEQ ID NO:1. The lengthof the oligonucleotide is not critical, as long as it is capable ofhybridizing to the target nucleic acid molecule. However, longer nucleicacid molecules are more difficult to prepare and require longerhybridization times. Therefore, the oligonucleotide should not be longerthan necessary. Accordingly, the oligonucleotide should contain at least10 nucleotides, preferably at least 15 nucleotides, and more preferablyat least 20 nucleotides. Normally, the oligonucleotide will not containmore than 60 nucleotides, preferably not more than 30 nucleotides, andmore preferably not more than 25 nucleotides. Such oligonucleotides maybe used as described herein as primers for DNA synthesis (e.g., asprimers in PCR; “amplimers”), as probes for detecting the presence oftarget DNA in a sample (e.g., northern or Southern blots and in situhybridization), as therapeutic agents (e.g., in antisense therapy), orfor other purposes. Oligonucleotides may be single- or double-stranded,with the double-stranded forms having one or both ends blunt or stepped.

[0050] The oligonucleotides may be obtained or derived by known methodsfrom natural sources. Alternatively, the oligonucleotides may beproduced synthetically according to methods known in the art. Suchmethods include, for example, cloning and restriction of appropriatesequences or direct chemical synthesis by any suitable method. Variouschemical methods for making oligonucleotides are known in the art,including the phosphotriester method, the phosphodiester method; thediethylphosphoramidite method; the solid support method, and theH-phosphonate method [for reviews, see Caruthers, Science 230:281-5(1985); Caruthers et al., Methods Enzymol 211:3-20 (1992)]. Typically,preparation of oligonucleotides is carried out by automatedphosphoramidite synthesis on polymer support. Nucleic acid moleculesconsisting of 100 or more nucleotides may also be produced by suchmethods.

[0051] The hparp2 polynucleotides of the invention include variants,which are polynucleotides that encode hPARP2 or a functional equivalentthereof, and which can include deletions, insertions, or substitutionsof nucleotide residues. As used herein a “deletion” is a change in anucleotide or amino acid sequence in which one or more nucleotides oramino acid residues, respectively, are absent. As used herein an“insertion” or “addition” is a change in a nucleotide or amino acidsequence that results in the addition of one or more nucleotides oramino acid residues, respectively. As used herein a “substitution” is achange in a nucleotide or amino acid sequence in which one or morenucleotides or amino acids are replaced by different nucleotides oramino acids, respectively.

[0052] Polynucleotide variants also included within the scope of thepresent invention are alleles or alternative naturally occurring formsof hparp2. Alleles result from naturally occurring mutations, i.e.,deletions, insertions or substitutions, in the genomic nucleotidesequence, which may or may not alter the structure or function or theexpressed polypeptides. Each of these types of mutational changes mayoccur alone, or in combination with the others, one or more times in agiven allelic sequence. Single nucleotide polymorphisms (SNPs) mayoccur, in which a single base mutation may define an alteredpolypeptide, which in turn may be associated with an overt phenotypicdifference. Of course, SNPs may be silent, as they may not change theencoded polypeptide, or any change they do encode may have no effect onphenotype.

[0053] The invention further embraces natural homologs of the humanparp2 DNA that occur in other animal species, preferably other mammalspecies, and more preferably other primate species. Such specieshomologs, in general, share significant homology at the nucleotide levelwithin the protein-coding regions. Thus, the invention encompassespolynucleotides that share at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, or at least 99% nucleotideidentity with the protein-coding region of a polynucleotide encoding ahuman hPARP2 polypeptide, e.g., the polynucleotide defined by SEQ IDNO:1. Percent sequence “homology” with respect to polynucleotides of theinvention can be defined as the percentage of nucleotide bases in acandidate sequence that are identical to nucleotides in thehPARP2-encoding sequence after aligning the sequences and introducinggaps, if necessary, to achieve maximum percent sequence identity.Computer software is available (from commercial and public domainsources) for calculating percent identity in an automated fashion (e.g.,FASTA).

[0054] The invention includes polynucleotides that have been engineeredto selectively modify the cloning, processing, and/or expression of thehPARP2 gene product. Mutations may be introduced using techniques wellknown in the art, e.g., site-directed mutagenesis to insert newrestriction sites, to alter glycosylation patterns, or to change codonpreferences inherent in the use of certain expression systems, whilesimultaneously maintaining control of the amino acid sequence of theexpressed polypeptide product. For example, codons preferred by aparticular prokaryotic or eukaryotic host cell can be selected toincrease the rate of hPARP2 expression or to produce recombinant RNAtranscripts having desirable properties, such as longer half-lives.

[0055] The hparp2 polynucleotides can be synthesized, wholly or partly,using chemical methods well known in the art. “Chemically synthesized,”as used herein and is understood in the art, refers to purely chemical,as opposed to enzymatic, methods for producing polynucleotides. “Wholly”chemically synthesized DNA sequences are therefore produced entirely bychemical means; “partly” chemically synthesized DNAs embrace thosewherein only portions of the resulting DNA were produced by chemicalmeans.

[0056] DNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences of the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiester linkages within the backbone of the molecule.

[0057] The invention also provides hPARP2 peptide nucleic acid (PNA)molecules. These hPARP2 PNAs are informational molecules that have aneutral “peptide-like” backbone with nucleobases that allow themolecules to hybridize to complementary hPARP2-encoding DNA or RNA withhigher affinity and specificity than corresponding oligonucleotides(PerSeptive Biosystems).

[0058] Polypeptide Expression Systems

[0059] Knowledge of hPARP2-encoding DNA sequences enables the artisan tomodify cells to permit or increase expression of hPARP2. Accordingly,host cells are provided, including prokaryotic or eukaryotic cells,either stably or transiently modified by introduction of apolynucleotide of the invention to permit expression of the encodedhPARP2 polypeptide. Autonomously replicating recombinant expressionconstructs such as plasmid and viral DNA vectors incorporatinghPARP2-encoding sequences are also provided.

[0060] Expression constricts are also provided comprisinghPARP2-encoding polynucleotides operatively linked to an endogenous orexogenous expression control DNA sequence and a transcriptionterminator. Expression control DNA sequences include promoters,enhancers, and operators, and are generally selected based on theexpression systems in which the expression construct is to be used.Preferred promoter and enhancer sequences are generally selected for theability to increase gene expression, while operator sequences aregenerally selected for the ability to regulate gene expression.Preferred constructs of the invention also include sequences necessaryfor replication in a host cell. Expression constructs are preferablyused for production of an encoded hPARP2 polypeptide, but may also beused to amplify the construct itself.

[0061] Polynucleotides of the invention may be introduced into the hostcell as part of a circular plasmid, or as linear DNA comprising anisolated protein coding region or a viral vector. Methods forintroducing DNA in to a host cell include transformation, transfection,electroporation, nuclear injection, or fusion with carriers such asliposomes, micelles, ghost cells, and protoplasts. Expression systems ofthe invention include, for example, bacteria, yeast, fungal, plant,insect, invertebrate, amphibian, and mammalian cell systems. Somesuitable prokaryotic host cells include, for example, E. coli strainsSG-936, HB 101, W3110, X1776, X2282, DHI, and MRC1, Pseudomonas species,Bacillus species such as B. subtilis, and Streptomyces species. Suitableeukaryotic host cells include yeasts, such as Saccharomyces cerevisiae,S. pombe, Pichia pastoris and other fungi, insect cells such as sf9 orsf21 cells (Spodoptera frugiperda), animal cells such as Chinese hamsterovary (CHO) cells, human cells such as JY, 293, and NIH3T3 cells, andplant cells such as Arabidopsis thaliana cells. The hparp2 nucleotidesequence, or any portion of it, may be cloned into a vector for theproduction of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of labeled nucleotides and an appropriate RNApolymerase such as T7, T3, or SP6.

[0062] The type of host cell, the form of the expressed hPARP2 product,the conditions of growth, etc., can be selected by the skilled artisanaccording to known criteria. Use of mammalian host cells is expected toprovide for such post-translational modifications (e.g., glycosylation,truncation, lipidation, and phosphorylation) as may be needed to conferoptimal biological activity on recombinant expression products of theinvention. Glycosylated and non-glycosylated forms of hPARP2polypeptides are embraced. The protein produced by a recombinant cellmay be secreted or may be contained intracellularly, depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing hparp2 can be designed withsignal sequences that direct secretion of hPARP2 through a particularprokaryotic or eukaryotic cell membrane.

[0063] Expression constructs may include sequences that facilitate, andpreferably promote, homologous recombination in a host cell. This can beaccomplished by replacing all or part of the naturally occurring hparp2promoter with all or part of a heterologous promoter so that the cellsexpress hPARP2 at higher levels. The heterologous promoter should beinserted so that it is operatively linked to hPARP2-encoding sequences[see, for example, PCT International Publication Nos. WO 94/12650, WO92/20808, and WO 91/09955].

[0064] Host cells of the invention are useful in methods for large-scaleproduction of hPARP2 polypeptide products. For example, host cells ofthe invention are a valuable source of immunogen for development ofantibodies that are immunoreactive with hPARP2 polypeptides. As anotherexample, recombinant hPARP2 can be produced and isolate from host cellsfor use in in vitro binding assays such as drug screening assays. Insuch methods, the host cells are grown in a suitable culture medium andthe desired polypeptide product is isolated from the cells or from themedium in which the cells are grown.

[0065] The polypeptide product can be isolated by purification methodsknown in the art, such as conventional chromatographic methods includingimmunoaffinity chromatography, receptor affinity chromatography,hydrophobic interaction chromatography, lectin affinity chromatography,size exclusion filtration, cation or anion exchange chromatography, highperformance liquid chromatography (HPLC), reverse phase HPLC, and thelike.

[0066] Still other methods of purification include those in which thedesired protein is expressed and purified as a fusion protein in whichthe hPARP2 polypeptide is ligated to a heterologous amino acid sequence.Suitable heterologous sequences can include a specific tag, label, orchelating moiety that is recognized by a specific binding partner oragent. For example, for screening of peptide libraries for modulators ofhPARP2 activity, it is possible to express an hPARP2 protein fused to aselected heterologous protein selected to be specifically identifiableusing a probe antibody. A fusion protein may also be engineered tocontain a cleavage site (e.g., a factor XA or enterokinase sensitivesequence) located between the hPARP2 sequence and the heterologousprotein sequence, to permit the hPARP2 protein to be cleaved from theheterologous protein and subsequently purified. Cleavage of the fusioncomponent may produce a form of the desired protein having additionalamino acid residues resulting from the cleavage process.

[0067] Exemplary heterologous peptide domains include metal-chelatingpeptides such as histidine-tryptophan modules that allow purification onimmobilized metals [Porath, Protein Expr Purif 3:263-81 (1992)], andprotein A domains that allow purification on immobilized immunoglobulin.Another useful system is the divalent cation-binding domain andantibodies specific thereto used in the peptide extension/immunoaffinitypurification system described in U.S. Pat. Nos. 4,703,004; 4,782,137;4,851,431; and 5,011,912. This system is commercially available as theFLAG® system from Immunex Corp. (Seattle, Wash.). Another suitableheterologous fusion partner is glutathione S-transferase (GST), whichcan be affinity purified using immobilized glutathione. Other usefulfusion partners include immunoglobulins and fragments thereof, e.g., Fcfragments.

[0068] Identification of host cells expressing recombinant hPARP2 may becrucial to identifying appropriate expression systems. Accordingly,expression constructs of the invention may also include sequencesencoding one or more selectable markers that permit identification ofhost cells bearing the construct in operative condition. It is alsocontemplated that, in addition to the insertion of heterologous promoterDNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctionalCAD gene that encodes carbamyl phosphate synthase, aspartatetranscarbamylase, and dihydroorotase) and/or intron DNA may be insertedalong with the heterologous promoter DNA. If linked to thehPARP2-encoding sequence, amplification of the marker DNA by standardselection methods results in co-amplification of the hPARP2-encodingsequences in the cells. Detection of expression of the marker gene inresponse to induction or selection usually indicates expression ofhPARP2 as well. Alternatively, if the hparp2 polynucleotide is insertedwithin a marker gene sequence, recombinant cells containing hparp2 canbe identified by the absence of marker gene function.

[0069] Host cells that contain the coding sequence for hPARP2 andexpress hPARP2 may be identified by a variety of other procedures knownto those of skill in the art. These procedures include, but are notlimited to, DNA-DNA or DNA-RNA hybridization and protein bioassay orimmunoassay techniques that include membrane-based, solution-based, orchip-based technologies for the detection and/or quantification of thenucleic acid or protein.

[0070] The presence of the hparp2 polynucleotide sequence can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingfragments of hparp2 disclosed in SEQ ID NO:1 as probes. Nucleic acidamplification based assays involve the use of oligonucleotides based onthe hparp2 sequence to detect transformants containing hparp2 DNA orRNA. Labeled hybridization or PCR probes for detecting hparp2polynucleotide sequences can be made by various methods, includingoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide.

[0071] In an embodiment of the present invention, hPARP2 or a variantthereof and/or a host cell line that expresses the hPARP2 or variantthereof may be used to screen for antibodies, peptides, or othermolecules, such as organic or inorganic molecules, that act asmodulators of a biological or immunological activity of hPARP2. Forexample, anti-hPARP2 antibodies capable of neutralizing the polymeraseor DNA-binding activity of hPARP2 may be used to inhibit hPARP2-mediatedcell death. Alternatively, screening of peptide libraries or organiclibraries made by combinatorial chemistry with recombinantly expressedhPARP2 or variants thereof or cell lines expressing hPARP2 or variantsthereof may be useful for identification of therapeutic molecules thatfunction by modulating a biological or immunological activity of hPARP2.Synthetic compounds, natural products, and other sources of potentiallybiologically active material can be screened in a number of ways deemedroutine by those of skill in the art. For example, nucleotide sequencesencoding the DNA-binding domain of hPARP2 may be expressed in a hostcell, which can be used for screening of allosteric modulators, eitheragonists or antagonists, of hPARP2 activity. Alternatively, nucleotidesequences encoding the conserved catalytic domain of hPARP2 can beexpressed in host cells and used to screen for inhibitors of ADP-ribosepolymerization.

[0072] hPARP2 Polypeptides

[0073] The invention also provides purified and isolated mammalianhPARP2 polypeptides. An exemplary hPARP2 polypeptide has an amino acidsequence defined in SEQ ID NO:2. hPARP2 polypeptides of the inventionmay be isolated from natural cell sources or may be chemicallysynthesized, but are preferably produced by recombinant proceduresinvolving host cells of the invention. hPARP2 products of the inventionmay be full-length polypeptides, or variant polypeptide products such asfragments, truncates, deletion mutants, and other variants thereof thatretain specific hPARP2 biological activity. As used herein,“biologically active” refers to an hPARP2 polypeptide having structural,regulatory or biochemical functions of the naturally occurring hPARP2protein. Specifically, an hPARP2 protein of the present invention hasthe ability to bind DNA and to polymerize ADP-ribose subunits inresponse to DNA damage in a cell.

[0074] The protein and fragments of the present invention may beprepared by methods known in the art. Such methods include isolating theprotein directly from cells, isolating or synthesizing DNA encoding theprotein and using the DNA to produce recombinant protein, andsynthesizing the protein chemically from individual amino acids.

[0075] The hPARP2 polypeptides can be isolated from a biological sample,such as a solubilized cell fraction, by standard methods. Some suitablemethods include precipitation and liquid chromatographic protocols suchas ion exchange, hydrophobic interaction, and gel filtration [see, e.g.,Deutscher (Ed.), Methods Enzymol (Guide to Protein Chemistry, SectionVII) 182:309 (1990); Scopes, Protein Purification, Springer-Verlag, NewYork (1987)]. Alternatively, purified material is obtained by separatingthe protein on preparative SDS-PAGE gels, slicing out the band ofinterest and electrocluting the protein from the polyacrylamide matrixby methods known in the art. The detergent SDS is removed from theprotein by known methods, such as by dialysis or the use of a suitablecolumn, such as the Extracti-Gel® column (Pierce).

[0076] The hPARP2 polypeptide of the invention may also be chemicallysynthesized, wholly or partly, by methods known in the art. Suitablemethods for synthesizing the protein are described in the art [e.g.,Stuart and Young, Solid Phase Peptide Synthesis, 2d ed., Pierce ChemicalCo. (1984)]. For example, peptides can be synthesized by solid phasetechniques, cleaved from the resin, and purified by preparative highperformance liquid chromatography (HPLC) [see, e.g., Roberge et al.,Science 269:202-4 (1995)]. Automated synthesis may be accomplished, forexample, using the ABI 431 A Peptide Synthesizer (Perkin Elmer, Norwalk,Conn.) in accordance with the instructions provided by the manufacturer.The composition of the synthetic peptides may be confirmed by amino acidanalysis or sequencing (e.g., the Edman degradation procedure).

[0077] As described in greater detail above, recombinant hPARP2 proteinmay be produced in and isolated from a host cell transformed with anexpression vector containing an hparp2 nucleotide sequence and grown incell culture. As described herein, the host cells, either prokaryotic oreukaryotic, are either stably or transiently transfected (eukaryotic) ortransformed (prokaryotic) with an hPARP2-encoding polynucleotide of theinvention in a manner that permits directed expression of an hPARP2polypeptide. In such methods, the host cells are grown in a suitableculture medium and the desired polypeptide products are isolated fromthe cells or from the medium in which the cells are grown. Isolation ofthe polypeptides can be accomplished by, for example, immunoaffinitypurification. The use of transformed host cells is preferred forlarge-scale production of hPARP2 polypeptides.

[0078] The invention includes polypeptides comprising amino acidsequences that are substantially homologous to the sequences of hPARP2polypeptides described herein. For example, the invention includespolypeptides whose corresponding amino acid sequences have at least 90%,preferably at least 95%, more preferably at least 98%, and still morepreferably at least 99% identity with the polypeptide sequence definedin SEQ ID NO:2.

[0079] Percent sequence “identity” with respect to a preferredpolypeptide of the invention can be defined as the percentage of aminoacid residues in a candidate sequence that are identical to amino acidresidues in the reference hPARP2 sequence after aligning the sequencesand introducing gaps, if necessary, to achieve maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity.

[0080] Percent sequence “homology” with respect to a preferredpolypeptide of the invention can be defined as the percentage of aminoacid residues in a candidate sequence that are identical to amino acidresidues in the reference hPARP2 sequence after aligning the sequencesand introducing gaps, if necessary, to achieve maximum percent sequenceidentity, and also considering any conservative substitutions as part ofthe sequence identity.

[0081] Determinations of whether two amino acid sequences aresubstantially homologous can also be based on FASTA searches [Pearson etal., Proc Natl Acad Sci USA 85:2444-8 (1988)]. Alternatively, percenthomology is calculated as the percentage of amino acid residues in thesmaller of the two sequences that align with identical amino acidresidues in the sequence being compared, when four gaps in a length of100 amino acids may be introduced to maximize alignment. See Dayhoff, inAtlas of Protein Sequence and Structure, Vol. 5, National BiochemicalResearch Foundation, Washington, D.C. (1972), at p. 124.

[0082] A polypeptide may be considered homologous to an hPARP2polypeptide of the invention if polynucleotides encoding the twopolypeptides hybridize with one another. A higher degree of homology isshown if the hybridization occurs under hybridization conditions ofgreater stringency. Control of hybridization conditions and therelationships between hybridization conditions and degree of homologyare understood by those skilled in the art. See, e.g., Sambrook et al.(1989). Thus, a homologous polypeptide may be a polypeptide that isencoded by a polynucleotide that hybridizes with a polynucleotideencoding a polypeptide of the invention under hybridization conditionshaving a specified degree of stringency.

[0083] It may be desirable that such structurally homologouspolypeptides will also exhibit functional homology, insofar as thehomologous polypeptide has substantially homologous polypeptides may beconsidered functionally homologous if they exhibit similar binding of aligand, or similar immune reactivity, etc.

[0084] However, it is known that two polypeptides or two polynucleotidesmay be considered to be substantially homologous in structure, and yetdiffer substantially in function. For example, single nucleotidepolymorphisms (SNPs) among alleles may be expressed as polypeptideshaving substantial differences in function along one or more measurableparameters such as antibody- or ligand-binding affinity or enzymaticsubstrate specificity, and the like. Other structural differences, suchas substitutions, deletions, splicing variants, and the like, may affectthe function of otherwise structurally identical or homologouspolypeptides.

[0085] The hPARP2 polypeptides of the invention include functionalderivatives of the hPARP2 polypeptide defined in SEQ ID NO:2. Suchfunctional derivatives include polypeptide products that possesses astructural feature or a biological activity that is substantiallysimilar to a structural feature or a biological activity of the hPARP2protein. Accordingly, functional derivatives include variants,fragments, and chemical derivatives of the parent hPARP2 protein.

[0086] As used herein “variant” refers to a molecule substantiallysimilar in structure and function to either the entire hPARP2 molecule,or to a fragment thereof. A molecule is said to be “substantiallysimilar” to another if both molecules have substantially similarstructures or if both molecules possess a similar biological activity.Thus, provided that two molecules possess a similar activity, they areconsidered variants, as that term is used herein, even if one of themolecules possesses a structure not found in the other molecule, or ifthe sequence of amino acid residues is not identical.

[0087] Among the variant polypeptides provided under the invention arevariants that comprise one or more changes in the amino acid sequence ofthe hPARP2 polypeptide. Such sequence-based changes include deletions,substitutions, or insertions in the hPARP2 sequence, as well ascombinations thereof.

[0088] Deletion variants of the hPARP2 polypeptides are polypeptides inwhich at least one amino acid residue of the sequence is removed.Deletions can be effected at one or both termini of the protein, or withremoval of one or more residues within the hPARP2 amino acid sequence.Deletion variants include, for example, all incomplete fragments of thehPARP2 polypeptides of the invention. As used herein “fragment” refersto any polypeptide subset of the hPARP2 protein. A preferred fragment isa purified and isolated hPARP2 polypeptide comprising amino acids 1-49of the amino acid sequence defined in SEQ ID NO:2. Alternative preferredfragments include purified and isolated hPARP2 polypeptides comprisingat least one amino acid residue from a region consisting of amino acids1-49 of the amino acid sequence defined in SEQ ID NO:2. Such fragmentsinclude, for example, fragments comprising amino acids 49-583 of theamino acid sequence defined by SEQ ID NO:2, as well as N-terminallyextended fragments of that sequence and C-terminal truncates thereof.

[0089] Fragments of bPARP2 that exhibit a biological activitycharacteristic of hPARP2 and that are soluble (i.e., not membrane bound)are desirable. A soluble fragment is preferably generated by deletingany membrane-spanning region(s) of the parent molecule or by deleting orsubstituting hydrophilic amino acid residues for hydrophobic residues.Identification of such residues is well known in the art.

[0090] Substitution variants are provided, including polypeptides inwhich at least one amino acid residue of an hPARP2 polypeptide isreplaced by an alternative residue. Any substitution can be made, withconservative substitutions being preferred. Directed amino acidsubstitutions may be made based on well defined physicochemicalparameters of the canonical and other amino acids (e.g., the size,shape, polarity, charge, hydrogen-bonding capacity, solubility, chemicalreactivity, hydrophobicity, hydrophilicity, or the amphipathic characterof the residues.) as well as their contribution to secondary andtertiary protein structure. Substitution variants can includepolypeptides comprising one or more conservative amino acidsubstitutions, i.e., a substitution of one amino acid by another havingsimilar physicochemical character as desired. To illustrate, thecanonical amino acids can be grouped according to the followingcategories: Aliphatic Side Chains Gly, Ala; Val, Leu, Ile Aromatic SideChains Phe, Tyr, Trp Aliphatic Hydroxyl Side Chains Ser, Thr Basic SideChains Lys, Arg, His Acidic Side Chains Asp, Glu Amide Side Chains Asn,Gln Sulfur-Containing Side Chains Cys, Met Secondary Amino Group Pro

[0091] Substitutions are preferably made in accordance with thefollowing Table 1 when it is desired to controllably define thecharacteristics of the hPARP2 molecule. TABLE 1 Exemplary ConservativeOriginal Residue Substitutions Ala gly; ser Arg lys Asn gln; his Asp gluCys ser Gln asn Glu asp Gly ala; pro His asn; gln Ile leu; val Leu ile;val Lys arg; gln; glu Met leu; tyr; ile Phe met; leu; tyr Ser thr Thrser Trp tyr Tyr trp; phe Val ile; leu

[0092] Substantial changes in functional or immunological identity aremade by selecting substitutions that are more progressive than those inTable 1, i.e., selecting residues that differ more significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. The substitutionsthat are in general more progressive are those in which: (a) glycineand/or proline is substituted by another amino acid or is deleted orinserted; (b) a hydrophilic residue is substituted for a hydrophobicresidue; (c) a cysteine residue is substituted for (or by) any otherresidue; (d) a residue having an electropositive side chain issubstituted for (or by) a residue having an electronegative charge; or(e) a residue having a bulky side chain is substituted for (or by) onenot having such a side chain. Most preferred are amino acidsubstitutions that affect the solubility of hPARP2. These are mostpreferably generated by substituting hydrophilic for hydrophobic aminoacids.

[0093] Substitution variants, however, can include non-canonical ornon-naturally occurring amino acid residues substituted for amino acidresidues in the principal sequence. Substitution variants include thosepolypeptides in which amino acid substitutions have been introduced bymodification of polynucleotides encoding an hPARP2 polypeptide.

[0094] Insertion variants are provided, in which at least one amino acidresidue is present in addition to an hPARP2 amino acid sequence.Insertions may be located at either or both termini of the polypeptide,or may be positioned within the hPARP2 amino acid sequence. Insertionalvariants also include fusion proteins in which the amino or carboxyterminus of the hPARP2 polypeptide is fused to another polypeptide.Examples of such fusion proteins include immunogenic polypeptides,proteins with long circulating half-life (e.g., immunoglobulin constantregions), marker proteins (e.g., green fluorescent protein) and proteinsor polypeptides that facilitate purification of the desired hPARP2polypeptide (e.g., FLAG® tags or polyhistidine sequences). Anotherexample of a terminal insertion is a fusion of a signal sequence,whether heterologous or homologous to the host cell, to the N-terminusof the molecule to facilitate the secretion of the derivative fromrecombinant hosts. Intrasequence insertions (i.e., insertions within anhPARP2 molecule sequence) may range generally from about 1 to 10residues, more preferably 1 to 5.

[0095] Polypeptide variants of the invention also include mature hPARP2products, i.e., hPARP2 products wherein leader or signal sequences areremoved, as well as products having additional amino terminal residues.hPARP2 products having an additional methionine residue at position −1(Met⁻¹-hPARP2) are contemplated, as are hPARP2 products havingadditional methionine and lysine residues at positions −2 and −1,respectively (Met⁻²-Lys⁻¹-hPARP2). Other such variants are particularlyuseful for recombinant protein production in bacterial host cells.

[0096] The invention also encompasses hPARP2 variants having additionalamino acid residues resulting from use of specific expression systems.For example, use of commercially available vectors that express adesired polypeptide as a glutathione-S-transferase (GST) fusion productyields the desired polypeptide having an additional glycine residue atposition −1 (Gly⁻¹-hPARP2) upon cleavage of the GST component from thedesired polypeptide. Variants that result from expression in othervector systems are also contemplated.

[0097] The invention further provides hPARP2 polypeptide products thatare chemical derivatives of the hPARP2 polypeptide defined in SEQ IDNO:2. As used herein, the term “chemical derivative” refers to moleculesthat contain additional chemical moieties that are not normally a partof the naturally occurring molecule. Such moieties may impart desirableproperties to the derivative molecule, such as increased solubility,absorption, biological half-life, etc. The moieties may alternativelydecrease the toxicity of the derivative molecule, or eliminate orattenuate any undesirable side effect of the derivative molecule. Thus,chemical derivatives of hPARP2 polypeptides include polypeptides bearingmodifications other than (or in addition to) insertion, deletion orsubstitution of amino acid residues. Preferably, the modifications arecovalent in nature, and include, for example, chemical bonding withpolymers, lipids, non-naturally occurring amino acids, and other organicand inorganic moieties. Derivatives of the invention may be prepared toincrease circulating half-life of an hPARP2 polypeptide, or may bedesigned to improve targeting capacity for the polypeptide to desiredcells, tissues, or organs.

[0098] For example, methods are known in the art for modifying apolypeptide to include one or more water-soluble polymer attachmentssuch as polyethylene glycol, polyoxyethylene glycol, or polypropyleneglycol. Particularly preferred are hPARP2 products that have beencovalently modified with polyethylene glycol (PEG) subunits.Water-soluble polymers may be bonded at specific positions, for exampleat the amino terminus of the hPARP2 products, or randomly attached toone or more side chains of the polypeptide. Additional derivativesinclude hPARP2 species immobilized on a solid support, pinmicroparticle, or chromatographic resin, as well as hPARP2 speciesmodified to include one or more detectable labels, tags, chelatingagents, and the like.

[0099] Derivatization with bifunctional agents can be used to cross-linkTANK2 to a water-insoluble support matrix. Alternatively, reactivewater-insoluble matrices such as cyanogen bromide-activatedcarbohydrates and reactive substrates may be employed for proteinimmobilization [see, e.g., U.S. Pat. Nos. 3,969,287; 3,691,016;4,195,128; 4,247,642; 4,229,537; and 4,330,440.]

[0100] Expression of hPARP2 variants can be expected to have utility ininvestigating a biological activity characteristic of a wild-type hPARP2polypeptide. hPARP2 variants can be designed to retain all biological orimmunological properties characteristic for hPARP2, or to specificallydisable one or more particular biological or immunological properties ofhPARP2. For example, fragments and truncates may be designed to delete adomain associated with a particular property, or substitutions anddeletions may be designed to inactivate a property associated with aparticular domain. Forced expression (overexpression) of such variants(“dominant negative” mutants) can be employed to study the function ofthe protein in vivo by observing the phenotype associated with themutant.

[0101] Functional derivatives of hPARP2 having up to about 100 residuesmay be conveniently prepared by in vitro synthesis. If desired, suchfragments may be modified using methods known in the art by reactingtargeted amino acid residues of the purified or crude protein with anorganic derivatizing agent that is capable of reacting with selectedside chains or terminal residues. The resulting covalent derivatives maybe used to identify residues important for biological activity.

[0102] Functional derivatives of hPARP2 having altered amino acidsequences can also be prepared by mutating the DNA encoding hPARP2. Anycombination of amino acid deletion, insertion, and substitution may beemployed to generate the final construct, provided that the finalconstruct possesses the desired activity. Obviously, the mutations thatwill be made in the DNA encoding the functional derivative must notplace the sequence out of reading frame and preferably will not createcomplementary regions that could produce secondary mRNA structure [seeEP Patent Publication No. 75,444].

[0103] While the site for introducing a variation in the amino acidsequence is predetermined, the mutation per se need not bepredetermined. For example, to optimize the performance of a mutation ata given site, random mutagenesis, such as linker scanning mutagenesis,may be conducted at a target codon or target region to create a largenumber of derivative which could then be expressed and screened for theoptimal combination of desired activity. Alternatively, site-directedmutagenesis or other well-known technique may be employed to makemutations at predetermined sites in a DNA known sequence.

[0104] The technique of site-directed mutagenesis is well known in theart [e.g., Sambrook et al., supra, and McPherson (Ed.), DirectedMutagenesis: A Practical Approach, IRL Press, Oxford (1991)].Site-directed mutagenesis allows the production of hPARP2 functionalderivatives through use of specific oligonucleotide sequences thatencode the DNA sequence of the desired mutation. Site-directedmutagenesis methods and materials are commercially available, e.g., theQuikChange™ kit available from Stratagene (La Jolla, Calif.). One canselectively generate precise amino acid deletions, insertions, orsubstitutions using this method. Amino acid sequence deletions generallyrange from about 1 to 30 residues, more preferably 1 to 10 residues, andtypically are contiguous. The most preferred deletions are those thatare performed to generate catalytic fragments or DNA-binding fragments.

[0105] Mutations designed to increase the affinity of hPARP2 may beguided by the introduction of the amino acid residues that are presentat homologous positions in other poly(ADP-ribose) polymerase proteins.Similarly, such mutant hPARP2 molecules may be prepared that lackresidues of a functional domain, e.g., the catalytic domain, to create adominant negative protein.

[0106] It is difficult to predict a priori the exact effect anyparticular modification, e.g., substitution, deletion, insertion, etc.,will have on the biological activity of hPARP2. However, one skilled inthe art will appreciate that the effect will be evaluated by routinescreening assays. For example, a derivative typically is made by linkerscanning site-directed mutagenesis of the DNA encoding the native hPARP2molecule. The derivative is then expressed in a recombinant host, and,optionally, purified from the cell culture, for example, byimmunoaffinity chromatography. The activity of the cell lysate or thepurified derivative is then screened in a suitable screening assay forthe desired characteristic. For example, a change in the immunologicalcharacter of the functional derivative, such as affinity for a givenantibody, is measured by a competitive type immunoassay. Changes inother parameters of the expressed product may be measured by theappropriate assay.

[0107] Antibodies

[0108] The present invention provides antibodies that bind withspecificity to an hPARP2 polypeptide. An “antibody” as used herein isdefined broadly as a protein that characteristically immunoreacts withan epitope (antigenic determinant) that is characteristic of the hPARP2polypeptide. As used herein, an antibody is said to “immunoreact” withan antigen such as a polypeptide if the antibody specifically recognizesand binds an epitope that is characteristic of the antigen by way of oneor more variable regions or one or more of the complementaritydetermining regions (CDRs) of the antibody.

[0109] An antibody that is immunoreactive with a given polypeptide mayexhibit cross-reactivity to another polypeptide if the two polypeptideseach comprise a common structural feature that defines the samecharacteristic epitope. In the case of related polypeptides,cross-reactivity can correlate to common structural features such assequence identity, homology, or similarity found among the relatedpolypeptides. Accordingly, families of polypeptides can often beidentified by a cross-reactive antibody, i.e., an antibody thatimmunoreacts with some or all of the members of the polypeptide familysharing the common epitope. Thus, the invention encompasses antibodiesthat immunoreact with a particular member of the hPARP2 family ofpolypeptides, e.g., a polypeptide comprising the amino acid sequencedefined by SEQ ID NO:2 or comprising amino acid residues 1 to 49 of SEQID NO:2. The invention further encompasses antibodies that immunoreactwith some or all members of the hPARP2 family of polypeptides. Screeningassays to determine the binding specificity of an antibody are wellknown and routinely practiced in the art [see Harlow et al. (Eds.),Antibodies: A Laboratory Manual, Ch. 6, Cold Spring Harbor Laboratory,Cold Spring Harbor N.Y. (1988)]. The immunoreactive specificity withwhich an antibody binds to a given polypeptide antigen is to bedistinguished from interactions with other proteins, e.g.,Staphylococcus aureus protein A or other antibodies in ELISA techniques,that are mediated through parts of the antibody other than the variableregions, in particular the constant regions of the antibody.

[0110] Antibodies include, for example, monoclonal antibodies,polyclonal antibodies, single chain antibodies (scFv antibodies),chimeric antibodies, multifunctional/multispecific (e.g., bifunctionalor bispecific) antibodies, humanized antibodies, human antibodies, andCDR-grafted antibodies (including moieties that include CDR sequencesthat specifically immunoreact with a polypeptide of the invention).Antibodies according to the invention also include antibody fragments,so long as they exhibit the desired biological activity. “Antibodyfragments” comprise a portion of a full-length antibody, generally theantigen binding or variable region thereof. Examples of antibodyfragments include Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies;linear antibodies; single-chain antibody molecules; and multispecificantibodies formed from antibody fragments.

[0111] Antibodies of the invention can be produced by any method knownin the art. For example, polyclonal antibodies are isolated from mammalsthat have been immunized against the protein or a functional analog inaccordance with methods known in the art. Briefly, polyclonal antibodiesmay be produced by injecting an immunogenic hPARP2 polypeptide(immunogen) into a host mammal (e.g., rabbit, mouse, rat, or goat).Adjuvants may be employed to increase the immune response. Sera from thehost mammal are extracted and screened to obtain polyclonal antibodiesthat are specific for (immunoreact with) the hPARP2 polypeptide.

[0112] Monoclonal antibodies (also referred to herein as “mAbs”) arepreferred. As used herein “monoclonal antibody” refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific(“monospecific”), being directed against a single antigenic site.Furthermore, in contrast to conventional (polyclonal) antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen.

[0113] The modifier “monoclonal” indicates the character of the antibodyas being obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. Monoclonal antibodies may be preparedusing any suitable technique capable of yielding a continuous cell lineproducing a homogeneous antibody. Such methods include the immunologicalmethod [Kohler and Milstein, Nature 256:495-497 (1975); Campbell,“Monoclonal antibody technology. the production and characterization ofrodent and human hybridomas” in Burdon et al. (Eds.), LaboratoryTechniques in Biochemistry and Molecular Biology, Vol. 13, ElsevierScience Publishers, Amsterdam (1985)] or any similar method. Monoclonalantibodies may also be isolated from phage antibody libraries [Clacksonet al., Nature 352:624-8 (1991); Marks et al., J Mol Biol 222:581-97(1991)].

[0114] To illustrate, to produce monoclonal antibodies a host mammal isimmunized by injection of an immunogenic hPARP2 polypeptide, and thenboosted. Spleens are collected from immunized mammals a few days afterthe final boost. Cell suspensions from the spleens are fused with atumor cell line to create immortalized hybrid cell lines or“hybridomas.” Individual clones can be isolated by limiting dilution andthen tested for the specificity of the antibodies they produce. Selectedcells can then be grown, e.g., by the ascites method, to provide acontinuous source of the desired homogeneous antibody.

[0115] Antibodies can be engineered using genetic techniques to producechimeric antibodies including protein components from two or morespecies. For use in in vivo applications with a human subject, theantibody can be “humanized,” i.e., modified to contain an antigenbinding region from one species, e.g., a rodent, with the bulk of theantibody replaced with sequences derived from human immunoglobulin. Inone method, the non-human CDRs of one species e.g., a mouse or rabbit,are inserted into a framework sequence of another species, e.g., ahuman, or into a consensus framework sequence. Further changes can thenbe introduced into the antibody framework to modulate affinity orimmunogenicity of the engineered antibody. Methods are also known forinducing expression of engineered antibodies in various cell types, suchas mammalian and microbial cell types. Numerous techniques for preparingengineered antibodies are described in the art [e.g., Owens and Young, JImmunol Meth 168:149-65 (1994)].

[0116] Antibodies further include recombinant polyclonal or monoclonalFab fragments [e.g., Huse et al., Science 246:1275-81 (1989)].Alternatively, techniques described for the production of single chainantibodies (e.g., U.S. Pat. No. 4,946,778) can be adapted to producehPARP2-specific single chain antibodies (e.g., single chain Fvfragments; abbreviated “scFv”). Rapid, large-scale recombinant methodsfor generating antibodies may be employed such as phage display orribosome display methods, optionally followed by affinity maturation[see, e.g., Ouwehand et al., Vox Sang 74(Suppl 2):223-232 (1998); Raderet al., Proc Natl Acad Sci USA 95:8910-8915 (1998); Dall'Acqua et al.,Curr Opin Struct Biol 8:443-450 (1998)].

[0117] Fully human antibodies are especially preferred for therapeuticuse in humans, but they are typically difficult to produce. For example,when the immunogen is a human self-antigen, a human will typically notproduce any immune response to the antigen. Methods for making fullyhuman antibodies have been developed and are known in the art.Accordingly, fully human antibodies can be produced by using animmunogenic hPARP2 polypeptide to immunize an animal (e.g., mouse) thathas been transgenically modified to express at least a significantfraction of the human repertoire of immunoglobulin genes [see e.g.,Bruggemann et al., Immunol Today 17:391-7 (1996)].

[0118] As noted herein, host cells of the invention are a valuablesource of immunogen for development of antibodies specificallyimmunoreactive with hPARP2. To be useful as an immunogen for thepreparation of polyclonal or monoclonal antibodies, an hPARP2 peptidefragment must contain sufficient amino acid residues to define animmunogenic epitope. If the fragment is too short to be immunogenic perse, it may be conjugated to a carrier molecule. Suitable carriermolecules include, for example, keyhole limpet hemocyanin (KLH) andbovine serum albumin (BSA). Conjugation may be carried out by methodsknown in the art. One such method is to combine a cysteine residue ofthe fragment with a cysteine residue on the carrier molecule.

[0119] Antibodies of the invention are useful for therapeutic methods(by modulating activity of hPARP2), diagnostic methods (by detectinghPARP2 in a sample), as well as purification of hPARP2. The antibodiesare particularly useful for detecting and/or quantitating hPARP2expression in cells, tissues, organs, and lysates and extracts thereof,as well as in fluids such as serum, plasma, cerebrospinal fluid, urine,sputum, peritoneal fluid, pleural fluid, or bronchoalveolar lavagefluid. Kits comprising an antibody of the invention for any of thepurposes described herein are also contemplated. In general, a kit ofthe invention also includes a control antigen with which the antibodyimmunoreacts, and may further include other reagents, containers, andpackage inserts.

[0120] Further, the invention includes neutralizing antibodies, i.e.,antibodies that significantly inhibit or impair a biological activity ofthe proteins or functional analogs of the invention. In particular,neutralizing antibodies inhibit or impair the poly(ADP-ribose)polymerase activity of hPARP2. Neutralizing antibodies may be especiallydesirable for therapeutic and diagnostic applications.

[0121] Functional equivalents further include fragments of antibodiesthat have the same binding characteristics as, or that have bindingcharacteristics comparable to, those of the whole antibody. Suchfragments may contain one or both Fab fragments or the F(ab′)₂ fragment.Preferably, the antibody fragments contain all six complementdetermining regions (“CDRs”) of the whole antibody, although fragmentscontaining fewer than all of such regions, such as three, four, or fiveCDRs, may also be functional. Fragments may be prepared by methodsdescribed in the art [e.g., Lamoyi et al., J Immunol Meth 56:235-43(1983); Parham, J Immunol 131:2895-902 (1983)].

[0122] Moreover, specific binding proteins can be developed usingisolated or recombinant hPARP2 products, hPARP2 variants, or cellsexpressing such products. Binding proteins are useful for purifyinghPARP2 products and detection or quantification of hPARP2 products influid and tissue samples using known immunological procedures. Bindingproteins are also manifestly useful in modulating (i.e., blocking,inhibiting, or stimulating) biological activities of hPARP2polypeptides, especially those activities involved in signaltransduction. Thus, neutralizing antibodies that inhibit the activity ofhPARP2 polypeptides are provided. Anti-idiotypic antibodies specific foranti-hPARP2 antibodies are also contemplated.

[0123] Detectable Polynucleotide and Polypeptide Probes

[0124] The present invention further provides a method of detecting thepresence of an hPARP2-encoding polynucleotide or an hPARP2 polypeptidein a sample. The method involves use of a labeled probe that recognizesthe presence of a defined target in the sample. The probe may be anantibody that recognizes an hPARP2 polypeptide, or an oligonucleotidethat recognizes a polynucleotide encoding hPARP2 polypeptide.

[0125] The probes of the invention can be detectably labeled inaccordance with methods known in the art. In general, the probe can bemodified by attachment of a detectable label (reporter) moiety to theprobe, or a detectable probe can be manufactured with a detectable labelmoiety incorporated therein. The detectable label moiety can be anydetectable moiety, many of which are known in the art, includingradioactive atoms, electron dense atoms, enzymes, chromogens and coloredcompounds, fluorogens and fluorescent compounds, members of specificbinding pairs, and the like.

[0126] Methods for labeling oligonucleotide probes have been describedin the art [see, e.g., Leary et al., Proc Natl Acad Sci USA 80:4045-49(1983); Renz and Kurz, Nucleic Acids Res 12:3435-44 (1984); Richardsonand Gum port, Nucleic Acids Res 11:6167-84 (1983); Smith et al., NucleicAcids Res 13:2399-412 (1985); Meinkoth and Wahl, Anal Biochem 138:267-84(1984); and U.S. Pat. Nos. 4,711,955; 4,687,732; 5,241,060; 5,244,787;5,328,824; 5,580,990; and 5,714,327].

[0127] Methods for labeling antibodies have been also been described[see, e.g., Hunter et al., Nature 144:495-6 (1962); David et al.,Biochemistry 13:1014-21 (1974); and U.S. Pat. Nos. 3,940,475 and3,645,090].

[0128] The label moiety may be radioactive. Some examples of usefulradioactive labels include ³²P, ¹²⁵I, ¹³¹I, and ³H. Use of radioactivelabels has been described [e.g., UK patent document 2,034,323 and U.S.Pat. Nos. 4,358,535 and 4,302,204].

[0129] Some examples of non-radioactive labels include enzymes,chromogens, atoms and molecules detectable by electron microscopy, andmetal ions detectable by their magnetic properties.

[0130] Some useful enzymatic labels include enzymes that cause adetectable change in a substrate. Some useful enzymes (and theirsubstrates) include, for example, horseradish peroxidase (pyrogallol ando-phenylenediamine), beta-galactosidase (fluoresceinbeta-D-galactopyranoside), and alkaline phosphatase(5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium). The useof enzymatic labels has been described in the art [see, e.g., UK patentdocument 2,019,404, European patent document EP 63,879, and Rotman, ProcNatl Acad Sci USA 47:1981-91 (1961)]

[0131] Useful reporter moieties include, for example, fluorescent,phosphorescent chemiluminescent, and bioluminescent molecules, as wellas dyes. Some specific colored or fluorescent compounds useful in thepresent invention include, for example, fluoresceins, coumarins,rhodamines, Texas red, phycoerythrins, umbelliferones, Luminol®, and thelike. Chromogens or fluorogens, i.e., molecules that can be modified(e.g., oxidized) to become colored or fluorescent or to change theircolor or emission spectra, are also capable of being incorporated intoprobes to act as reporter moieties under particular conditions.

[0132] The label moieties may be conjugated to the probe by methods thatare well known in the art. The label moieties may be directly attachedthrough a functional group on the probe. The probe either contains orcan be caused to contain such a functional group. Some examples ofsuitable functional groups include, for example, amino, carboxyl,sulfhydryl, maleimide, isocyanate, isothiocyanate.

[0133] Alternatively, label moieties such as enzymes and chromogens maybe conjugated to antibodies or nucleotides by means of coupling agents,such as dialdehydes, carbodiimides, dimaleimides, and the like.

[0134] The label moiety may also be conjugated to the probe by means ofa ligand attached to the probe by a method described above and areceptor for that ligand attached to the label moiety. Any of the knownligand-receptor binding pair combinations is suitable. Some suitableligand-receptor pairs include, for example, biotin-avidin or-streptavidin, and antibody-antigen. The biotin-streptavidin combinationmay be preferred.

[0135] Methods of Using hparp2 Polynucleotides and hPARP2 Polypeptides

[0136] The scientific value of the information contributed through thedisclosures of DNA and amino acid sequences of the present invention ismanifest. As one series of examples, knowledge of the sequence of a cDNAfor hparp2 makes possible through use of Southern hybridization orpolymerase chain reaction (PCR) the identification of genomic DNAsequences encoding hPARP2 and hPARP2 expression control regulatorysequences. DNA/DNA hybridization procedures carried out with DNAsequences of the invention under moderately to highly stringentconditions are also expected to allow the isolation of DNAs encodingallelic variants of hPARP2. Similarly, non-human species genes encodingproteins homologous to hPARP2 can also be identified by Southern and/orPCR analysis. As an alternative, complementation studies can be usefulfor identifying other human hPARP2 products as well as non-humanproteins, and DNAs encoding the proteins, sharing one or more biologicalproperties of hPARP2. Oligonucleotides of the invention are also usefulin hybridization assays to detect the capacity of cells to expresshPARP2. Polynucleotides of the invention may also be the basis fordiagnostic methods useful for identifying a genetic alteration in thehparp2 locus that underlies a disease state.

[0137] Oligonucleotides of the invention, as described herein, may beused in methods to amplify DNA for various purposes. “Amplification”according to the method of the invention refers to any molecular biologytechnique for detection of trace levels of a specific nucleic acidsequence by exponentially amplifying a template nucleic acid sequence.In particular, suitable amplification techniques include such techniquesas the polymerase chain reaction (PCR), the ligase chain reaction (LCR)and variants thereof. PCR is known to be a highly sensitive technique,and is in wide use [see, for example, Innis et al., PCR Protocols: AGuide to Methods and Applications, Academic Press, Inc., San Diego(1990); Dieffenbach and Dveksler, PCR Primer: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Plainview N.Y. (1995); and U.S. Pat.Nos. 4,683,195; 4,800,195; and 4,965,188]. LCR is more recentlydeveloped [Landegren et al., Science 241:1077-80 (1988) and Barany etal., PCR Methods and Applications 1:5-16 (1991); an LCR kit is availablefrom Stratagene]. LCR is known to be highly specific, and is capable ofdetecting point mutations. In certain circumstances, it is desirable tocouple the PCR and LCR techniques to improve precision of detection.Other amplification techniques may be employed in accordance to theinvention.

[0138] Oligonucleotide amplification primers are often provided asmatched pairs of single-stranded oligonucleotides; one with senseorientation (5′→3′) and one with antisense (3′←5′) orientation. Suchspecific primer pairs can be employed under optimized conditions foridentification of a specific gene or condition. Alternatively, the sameprimer pair, nested sets of oligomers, or even a degenerate pool ofoligomers, may be employed under less stringent conditions for detectionand/or quantitation of closely related DNA or RNA sequences.

[0139] Such oligonucleotides can be used in various methods known in theart to extend the specified nucleotide sequences. These methods permituse of a known sequence to determine unknown adjacent sequence, therebyenabling detection and determination of upstream sequences such aspromoters and regulatory elements.

[0140] For example, restriction-site polymerase chain reaction is adirect method that uses universal primers to retrieve unknown sequenceadjacent to a known locus [see, e.g., Gobinda et al., PCR Methods Applic2:318-22 (1993)]. In this method, genomic DNA is first amplified in thepresence of primer to a linker sequence and a primer specific to theknown region. The amplified sequences are subjected to a second round ofPCR with the same linker primer and another specific primer internal tothe first one. Products of each round of PCR are transcribed with anappropriate RNA polymerase and sequenced using reverse transcriptase.

[0141] Inverse PCR can be used to amplify or extend sequences usingdivergent primers based on a known region [Triglia et al., Nucleic AcidsRes 16:8186 (1988)]. The primers may be designed using Oligo 4.0(National Biosciences, Inc., Plymouth Minn.), or another appropriateprogram, to be 22-30 nucleotides in length, to have a GC content of 50%or more, and to anneal to the target sequence at temperatures about68°-72° C. This method uses several restriction enzymes to generate asuitable fragment in the known region of a gene. The fragment is thencircularized by intermolecular ligation and used as a PCR template.

[0142] Capture PCR is a method for PCR amplification of DNA fragmentsadjacent to a known sequence in human and yeast artificial chromosome(YAC) DNA [Lagerstrom et al., PCR Methods Applic 1:111-9 (1991)].Capture PCR also requires multiple restriction enzyme digestions andligations to place an engineered double-stranded sequence into anunknown portion of the DNA molecule before PCR. Walking PCR is a methodfor targeted gene walking that permits retrieval of unknown sequence[Parker et al., Nucleic Acids Res 19:3055-60 (1991)]. ThePromoterFinder™ kit (Clontech, Palo Alto, Calif.) uses PCR, nestedprimers, and special libraries to “walk in” genomic DNA. This processavoids the need to screen libraries and is useful in finding intron/exonjunctions.

[0143] Such methods can be used to explore genomic libraries to extend5′ sequence and to obtain endogenous hparp2 genomic sequence, includingelements such as promoters, introns, operators, enhancers, repressors,and the like. Preferred libraries for screening for full-length cDNAsare ones that have been size-selected to include larger cDNAs. Inaddition, randomly primed libraries are preferred in that they willcontain more sequences that contain the 5′ and upstream regions ofgenes.

[0144] The oligonucleotide probes may also be used for mapping theendogenous genomic sequence. The sequence may be mapped to a particularchromosome or to a specific region of the chromosome using well knowntechniques. These include in situ hybridization to chromosomal spreads[Venna et al., Human Chromosomes: A Manual of Basic Technique, PergamonPress, New York (1988)], flow-sorted chromosomal preparations, orartificial chromosome constructions such as YACs, bacterial artificialchromosomes (BACs), bacterial PI constructions, or single chromosomecDNA libraries.

[0145] Hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers are invaluable in extending genetic maps. Examples of geneticmaps can be found in Science 270:410 f (1995) and Science 265:1981f(1994). Often the placement of a gene on the chromosome of anothermammalian species may reveal associated markers even if the number orarm of a particular human chromosome is not known. Such sequences can beassigned to particular structural features of chromosomes by physicalmapping. This provides valuable information to investigators searchingfor disease genes using positional cloning or other gene discoverytechniques. Once a disease or syndrome has been crudely localized bygenetic linkage to a particular genomic region, any sequences mapping tothat area may represent associated or regulatory genes for furtherinvestigation [see, e.g., Gatti et al., Nature 336:577-80 (1988)]. Thepolynucleotides of the invention may also be used to detect differencesin the chromosomal location due to translocation, inversion, etc.,between normal, carrier, or affected individuals.

[0146] The DNA sequence information provided by the present inventionalso makes possible the development, e.g., through homologousrecombination or “knock-out” strategies [Capecchi, Science 244:1288-92(1989)], of animals that fail to express functional hPARP2 or thatexpress a variant of hPARP2. Such animals are useful as models forstudying the in vivo activities of hPARP2 and modulators thereof.

[0147] As described herein, the invention provides antisense nucleicacid sequences that recognize and hybridize to polynucleotides encodinghPARP2. Modifications of gene expression can be obtained by designingantisense sequences to the control regions of the hparp2 gene, such asthe promoters, enhancers, and introns. Oligonucleotides derived from thetranscription initiation site, e.g., between −10 and +10 regions of theleader sequence, are preferred. Antisense RNA and DNA molecules may alsobe designed to block translation of mRNA by preventing the transcriptfrom binding to ribosomes. The worker of ordinary skill will appreciatethat antisense molecules of the invention include those thatspecifically recognize and hybridize to hparp2 DNA (as determined bysequence comparison of hparp2 DNA to DNA encoding other knownmolecules). The antisense molecules of the invention also include thosethat recognize and hybridize to DNA encoding other members of the hPARP2family of proteins. Antisense polynucleotides that hybridize to multipleDNAs encoding other members of the hPARP2 family of proteins are alsoidentifiable through sequence comparison to identify characteristic orsignature sequences for the family of hPARP2 proteins. Accordingly, suchantisense molecules preferably have at least 95%, more preferably atleast 98%, and still more preferably at least 99% identity to the targethparp2 sequence.

[0148] Antisense polynucleotides are particularly relevant to regulatingexpression of hPARP2 by those cells expressing hparp2 mRNA. Antisensepolynucleotides (preferably 10 to 20 bp oligonucleotides) capable ofspecifically binding to hparp2 expression control sequences or hparp2RNA are introduced into cells, e.g., by a viral vector or a colloidaldispersion system such as a liposome. The antisense oligonucleotidebinds to the hparp2 target nucleotide sequence in the cell and preventstranscription or translation of the target sequence. Phosphorothioateand methylphosphonate antisense oligonucleotides are specificallycontemplated for therapeutic use under the invention. The antisenseoligonucleotides may be further modified by poly-L-lysine, transferrinpolylysine, or cholesterol moieties at their 5′ ends [for a recentreview of antisense technology, see Delihas et al., Nature Biotechnology15:751-3 (1997)].

[0149] The invention further comprises methods to modulate hPARP2expression by means of ribozyme technology [for a review, see Gibson andShillitoe, Mol Biotechnol 7:125-37 (1997)]. Ribozyme technology can beused to inhibit translation of hparp2 mRNA in a sequence specific mannerthrough (i) the hybridization of a complementary RNA to a target mRNAand (ii) cleavage of the hybridized mRNA through endonuclease activityinherent to the complementary RNA. Ribozymes can be identified byempirical methods such as using complementary oligonucleotides inribonuclease protection assays, but more preferably are specificallydesigned based on scanning the target molecule for accessible ribozymecleavage sites [Bramlage et al., Trends Biotechnol 16:434-8 (1998)].Delivery of ribozymes to target cells can be accomplished using eitherexogenous or endogenous delivery techniques well known and practiced inthe art. Exogenous can include use of targeting liposomes or directlocal injection. Endogenous methods include use of viral vectors andnon-viral plasmids.

[0150] Ribozymes can specifically modulate expression of hPARP2 whendesigned to be complementary to regions unique to a polynucleotideencoding hPARP2. “Specifically modulate,” therefore is intended to meanthat ribozymes of the invention recognize only a polynucleotide encodinghPARP2. Similarly, ribozymes can be designed to modulate expression ofall or some of the hPARP2 family of proteins. Ribozymes of this type aredesigned to recognize nucleotide sequences conserved all or some of thepolynucleotides encoding the hPARP2 family members.

[0151] The invention further embraces methods to modulate transcriptionof hparp2 through use of oligonucleotide-directed triple helix formation(also known as Hogeboom base-pairing methodology) [for a review, seeLavrovsky et al., Biochem Mol Med 62:11-22 (1997)]. Triple helixformation is accomplished using sequence-specific oligonucleotides thathybridize to double stranded DNA in the major groove as defined in theWatson-Crick model. This triple helix hybridization compromises theability of the original double helix to open sufficiently for thebinding of polymerases, transcription factors, or regulatory molecules.Preferred target sequences for hybridization include promoter andenhancer regions to permit transcriptional regulation of hPARP2expression. Oligonucleotides that are capable of triple helix formationcan alternatively be coupled to DNA damaging agents, which can then beused for site-specific covalent modification of target DNA sequences.See Lavrovsky et al. supra.

[0152] Both antisense RNA and DNA molecules and ribozymes of theinvention may be prepared by any method known in the art for thesynthesis of RNA molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid-phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro or in vivo transcription of DNA sequences encoding the antisenseRNA molecule. Such DNA sequences may be incorporated into a variety ofvectors with suitable RNA polymerase promoters such as T7 or SP6.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly can be introduced into cell lines, cells, ortissues.

[0153] Mutations in a gene that result in loss of normal function of thegene product may underlie hPARP2-related disease states. The inventioncomprehends gene therapy to restore hPARP2 activity as indicated intreating those disease states characterized by a deficiency or absenceof poly(ADP-ribose) polymerase activity associated with the hPARP2enzyme. Delivery of functional hparp2 gene to appropriate cells iseffected ex vivo, in situ, or in vivo by use of vectors, and moreparticularly viral vectors (e.g., adenovirus, adeno-associated virus, orretrovirus), or ex vivo by use of physical DNA transfer methods (e.g.,liposomes or chemical treatments) [see, e.g., Anderson, Nature 392(6679Suppl):25-30 (1998)]. Alternatively, it is contemplated that in otherdisease states, preventing the expression or inhibiting the activity ofhPARP2 will be useful in treating those disease states. Antisensetherapy or gene therapy can be applied to negatively regulate theexpression of hPARP2.

[0154] The DNA and amino acid sequence information provided by thepresent invention also makes possible the systematic analysis of thestructure and function of hPARP2 proteins. DNA and amino acid sequenceinformation for hPARP2 also permits identification of molecules withwhich an hPARP2 polypeptide will interact. Agents that modulate (i.e.,increase, decrease, or block) hPARP2 activity may be identified byincubating a putative modulator with hPARP2 and determining the effectof the putative modulator on hPARP2 activity. The selectivity of acompound that modulates the activity of the hPARP2 can be evaluated bycomparing its activity on the hPARP2 to its activity on other proteins.Cell-based methods, such as dihybrid or trihybrid assays (to identifyDNAs encoding binding partners) and split hybrid assays (to identifyinhibitors of hPARP2 polypeptide interaction with a known bindingpartner), as well as in vitro methods, including assays in which anhPARP2 polypeptide, hparp2 polynucleotide, or a binding partner thereofis immobilized, and solution assays are contemplated under theinvention.

[0155] Selective modulators may include, for example, antibodies andother proteins or peptides that specifically bind to an hPARP2polypeptide or an hPARP2-encoding polynucleotide, oligonucleotides thatspecifically bind to hPARP2 polypeptides or hPARP2-encodingpolynucleotides, and other non-peptide compounds (e.g., isolated orsynthetic organic molecules) that specifically react with hPARP2polypeptides or hPARP2-encoding polynucleotides. Modulators also includecompounds as described above but which interact with a specific bindingpartner of hPARP2 polypeptides. Mutant forms of hPARP2, such as thosethat affect the biological activity or cellular location of thewild-type hPARP2, are also contemplated under the invention. Presentlypreferred targets for the development of selective modulators include,for example:

[0156] (1) cytoplasmic or transmembrane regions of hPARP2 polypeptidesthat contact other proteins and/or localize hPARP2 within a cell;

[0157] (2) extracellular regions of hPARP2 polypeptides that bindspecific binding partners;

[0158] (3) regions of the hPARP2 polypeptides that bind substrate;

[0159] (4) allosteric regulatory sites of the hPARP2 polypeptides;

[0160] (5) regions of the hPARP2 polypeptides that mediatemultimerization. Still other selective modulators include those thatrecognize specific regulatory or hPARP2-encoding nucleotide sequences.Modulators of hPARP2 activity may be therapeutically useful in treatmentof a wide range of diseases and physiological conditions in whichaberrant hPARP2 activity is involved.

[0161] An hPARP2-coding polynucleotide sequence may be used for thediagnosis of diseases resulting from or associated with hPARP2expression or activity. For example, polynucleotide sequences encodinghPARP2 may be used in hybridization or PCR assays of biological samples,e.g., samples or extracts of fluids or tissues from biopsies orautopsies, to detect abnormalities in hPARP2 expression. Suchqualitative or quantitative methods may include Southern or northernanalysis, dot blot, or other membrane-based technologies; PCRtechnologies; dipstick, pin or chip technologies; and ELISA or othermultiple-sample format technologies. These types of techniques are wellknown in the art and have been employed in commercially availablediagnostic kits.

[0162] Such assays may be tailored to evaluate the efficacy of aparticular therapeutic treatment regimen and may be used in animalstudies, in clinical trials, or in monitoring the treatment of anindividual patient. To provide a basis for the diagnosis of disease, anormal or standard profile for hPARP2 expression must be established.This is accomplished by combining a biological sample taken from anormal subject with an hparp2 polynucleotide, under conditions suitablefor hybridization or amplification. Standard hybridization may bequantified by comparing the values obtained for normal subjects with adilution series of positive controls run in the same experiment where aknown amount of a purified hparp2 polynucleotide is used. Standardvalues obtained from normal samples may be compared with values obtainedfrom samples from subjects potentially affected by a disorder or diseaserelated to hPARP2 expression. Deviation between standard and subjectvalues establishes the presence of the disease state. If disease isestablished, an existing therapeutic agent is administered, andtreatment profile or values may be generated. The assay may be repeatedon a regular basis to evaluate whether the values progress toward orreturn to the normal or standard pattern. Successive treatment profilesmay be used to show the efficacy of treatment over a period of severaldays or several months.

[0163] Anti-hPARP2 antibodies are useful for the diagnosis ofconditions, disorders, or diseases characterized by or associated withabnormal expression of an hPARP2 polypeptide. Diagnostic assays forhPARP2 polypeptides include methods that employ a labeled antibody todetect an hPARP2 polypeptide in a biological sample such as a bodyfluid, cells, tissues, sections, or extracts of such materials. Thepolypeptides and antibodies of the present invention may be used with orwithout modification. Preferably, the polypeptide or the antibody willbe labeled by linking them, either covalently or non-covalently, with adetectable label moiety as described herein.

[0164] Antibody-based methods for detecting the presence of hPARP2polypeptides in biological samples are enabled by virtue of the presentinvention. Assays for detecting the presence of proteins with antibodieshave been previously described, and follow known formats, such asenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), andfluorescence activated cell sorting (FACS) and flow cytometry, westernblots, sandwich assays, and the like. These formats are normally basedon incubating an antibody with a sample suspected of containing thehPARP2 protein and detecting the presence of a complex between theantibody and the protein. The antibody is labeled either before, during,or after the incubation step. The specific concentrations of antibodies,the temperature and time of incubation, as well as other such assayconditions, can be varied, depending upon various factors including theconcentration of antigen in the sample, the nature of the sample, etc.Those skilled in the art will be able to determine operative and optimalassay conditions for each determination by employing routineexperimentation [see, e.g., Hampton et al., Serological Methods: ALaboratory Manual, APS Press, St Paul, Minn. (1990)].

[0165] To provide a basis for the quantitation of hPARP2 protein in asample or for the diagnosis of disease, normal or standard values ofhPARP2 polypeptide expression must be established. This is accomplishedby combining body fluids or cell extracts taken from a normal sample orfrom normal subjects, either animal or human, with antibody to an hPARP2polypeptide. The amount of standard complex formation may be quantifiedby comparing it with a dilution series of positive controls where aknown amount of antibody is combined with known concentrations of apurified hPARP2 polypeptide. Then, standard values obtained from normalsamples may be compared with values obtained from samples from testsample, e.g., subjects potentially affected by a disorder or diseaserelated to an hPARP2 expression. Deviation between standard and testvalues establishes the presence of the disease state.

[0166] Methods for Identifying Modulators of hPARP2 Activity

[0167] The hPARP2 protein, as well as fragments thereof possessingbiological activity can be used for screening putative modulatorcompounds in any of a variety of drug screening techniques. The term“modulator” as used herein refers to a compound that acts as an agonistor as an antagonist of hPARP2 activity. Modulators according to theinvention include allosteric modulators of activity as well asinhibitors of activity. An “agonist” of hPARP2 is a compound thatenhances or increases the ability of hPARP2 to carry out any of itsbiological functions. An example of such an agonist is an agent thatincreases the ability of hPARP2 to bind to damaged DNA or to polymerizeADP-ribose. An “antagonist” of hPARP2 is a compound that diminishes orabolishes the ability of hPARP2 to carry out any of its biologicalfunctions. An example of such antagonists is an anti-hPARP2 antibody.

[0168] Accordingly, the invention provides a method for screening aplurality of test compounds for specific binding affinity with an hPARP2polypeptide, comprising providing a plurality of test compounds;combining an hPARP2 polypeptide with each of the plurality of testcompounds for a time sufficient to allow binding under suitableconditions; and detecting binding of the hPARP2 polypeptide to each ofthe plurality of test compounds, thereby identifying those testcompounds that specifically bind the hPARP2 polypeptide.

[0169] The present invention also provides a method of identifying amodulator of a biological activity of an hPARP2 polypeptide, comprisingthe steps of a) contacting the compound with an hPARP2 polypeptide, b)incubating the mixture of step a) with a substrate under conditionssuitable for the biological activity, c) measuring the amount of thebiological activity; and d) comparing the amount of biological activityof step c) with the amount of biological activity obtained with thehPARP2 polypeptide, incubated without the compound, thereby determiningwhether the compound stimulates or inhibits the biological activity. Inone embodiment of the method, the hPARP2 polypeptide is a fragment fromthe non-catalytic region of the hPARP2 and provides a method to identifyallosteric modulators of hPARP2. In another embodiment, the hPARP2polypeptide is a fragment from the catalytic region of hPARP2 andprovides a method to identify inhibitors of the biological activity.

[0170] Accordingly, the polypeptide employed in such methods may be freein solution, affixed to a solid support, displayed on a cell surface, orlocated intracellularly. The modulation of activity or the formation ofbinding complexes between the hPARP2 polypeptide and the agent beingtested may be measured. hPARP2 polypeptides are amenable to biochemicalor cell-based high throughput screening (HTS) assays according tomethods known and practiced in the art, including melanophore assaysystems to investigate receptor-ligand interactions, yeast-based assaysystems, and mammalian cell expression systems [for a review, seeJayawickreme and Kost, Curr Opin Biotechnol 8:629-34 (1997)]. Automatedand miniaturized HTS assays are also comprehended [e.g., Houston andBanks, Curr Opin Biotechnol 8:734-40 (1997)].

[0171] Such HTS assays are used to screen libraries of compounds toidentify particular compounds that exhibit a desired property. Anylibrary of compounds may be used, including chemical libraries, naturalproduct libraries, combinatorial libraries comprising random or designedoligopeptides, oligonucleotides, or other organic compounds.

[0172] Chemical libraries may contain known compounds, proprietarystructural analogs of known compounds, or compounds that are identifiedfrom natural product screening.

[0173] Natural product libraries are collections of materials isolatedfrom naturals sources, typically, microorganisms, animals, plants, ormarine organisms. Natural products are isolated from their sources byfermentation of microorganisms followed by isolation and extraction ofthe fermentation broths or by direct extraction from the microorganismsor tissues (plants or animal) themselves. Natural product librariesinclude polyketides, non-ribosomal peptides, and variants (includingnon-naturally occurring variants) thereof [for a review, see Cane etal., Science 282:63-8 (1998)].

[0174] Combinatorial libraries are composed of large numbers of relatedcompounds, such as peptides, oligonucleotides, or other organiccompounds as a mixture. Such compounds are relatively straightforward todesign and prepare by traditional automated synthesis protocols, PCR,cloning or proprietary synthetic methods. Of particular interest arepeptide and oligonucleotide combinatorial libraries.

[0175] Still other libraries of interest include peptide, protein,peptidomimetic, multiparallel synthetic collection, recombinatorial, andpolypeptide libraries [for a review of combinatorial chemistry andlibraries created thereby, see Myers, Curr Opin Biotechnol 8:701-7(1997)].

[0176] Once compounds have been identified that show activity asmodulators of hPARP2 function, a program of optimization can beundertaken in an effort to improve the potency and or selectivity of theactivity. This analysis of structure-activity relationships (SAR)typically involves of iterative series of selective modifications ofcompound structures and their correlation to biochemical or biologicalactivity. Families of related compounds can be designed that all exhibitthe desired activity, with certain members of the family potentiallyqualifying as therapeutic candidates.

[0177] The invention also encompasses the use of competitive drugscreening assays in which neutralizing antibodies capable of binding anhPARP2 polypeptide specifically compete with a test compound for bindingto the hPARP2 polypeptide. In this manner, the antibodies can be used todetect the presence of any compound, e.g., another peptide that sharesone or more antigenic determinants with the hPARP2 polypeptide.

[0178] Therapeutic Uses of hPARP2-Encoding Polynucleotides and hPARP2Polypeptides

[0179] The invention provides a method for inhibiting the expression oractivity of hPARP2 therapeutically or prophylactically. The methodcomprises administering an hPARP2 antagonist in an amount effective forinhibiting hPARP2 expression or activity. The invention thus provides amethod for treating tissue damage resulting from cell damage or deathdue to necrosis or apoptosis, comprising administering to the animal aneffective amount of a compound that inhibits hPARP2 activity. Thismethod may be employed in treating persons who are or may be subject toany condition whose symptoms or pathology is mediated by hPARP2expression or activity.

[0180] The method may further involve administering an antagonist ofanother poly(ADP-ribose) polymerase activity, such as activityassociated with the enzymes PARP1, tankyrase, and the like. ExemplaryPARP1 antagonists suitable for use in this embodiment include, forexample, the compounds described in Banasik et al., J Biol Chem267:1569-75 (1992); and in PCT patent publications WO 99/11623 and WO99/11649. Alternatively, the hPARP2 inhibitory method may entail use ofa compound that antagonizes both hPARP2 and another enzyme having PARPactivity.

[0181] “Treating” as used herein refers to preventing a disorder fromoccurring in an animal that may be predisposed to the disorder, but hasnot yet been diagnosed as having it; inhibiting the disorder, i.e.,arresting its development; relieving the disorder, i.e., causing itsregression, or ameliorating the disorder, i.e., reducing the severity ofsymptoms associated with the disorder. “Disorder” is intended toencompass medical disorders, diseases, conditions, syndromes, and thelike, without limitation.

[0182] In particular, the method of the invention may be employed totreat humans therapeutically or prophylactically who are or may subjectto an inflammatory disorder. One aspect of the present invention derivesfrom the ability of hPARP2 and its functional derivatives to interactwith damaged DNA and to signal or induce cell death. Without intendingto be bound by any one theory, it is believed that, because inflammationinvolves processes that may be injurious to DNA, and because hPARP2 mayinappropriately induce cell death under such conditions, antagonists ofhPARP2 may be used to suppress injury associated with inflammation.

[0183] “Inflammatory disorder” as used herein can refer to any disease,disorder, or syndrome in which an excessive or unregulated inflammatoryresponse leads to excessive inflammatory symptoms, host tissue damage,or loss of tissue function. “Inflammatory disorders” can also refer topathological states mediated by influx of leukocytes and or neutrophilchemotaxis.

[0184] “Inflammation” as used herein refers to a localized, protectiveresponse elicited by injury or destruction of tissues, which serves todestroy, dilute or wall off (sequester) both the injurious agent and theinjured tissue. Inflammation is notably associated with influx ofleukocytes and or neutrophil chemotaxis. Inflammation may result frominfection with pathogenic organisms and viruses and from noninfectiousmeans such as trauma or reperfusion following myocardial infarction orstroke, immune response to foreign antigen, and autoimmune responses.Accordingly, inflammatory disorders amenable to the invention encompassdisorders associated with reactions of the specific defense system aswell as with reactions of the non-specific defense system.

[0185] As used herein, the term “specific defense system” refers to thecomponent of the immune system that reacts to the presence of specificantigens. Examples of classical response to foreign antigens, autoimmunediseases, and delayed type hypersensitivity response mediated byT-cells. Chronic inflammatory diseases, the rejection of solidtransplanted tissue and organs, e.g., kidney and bone marrowtransplants, and graft versus host disease (GVHD), are further examplesof inflammatory reactions of the specific defense system.

[0186] The term “non-specific defense system” as used herein refers toinflammatory disorders that are mediated by leukocytes that areincapable of immunological memory (e.g., granulocytes, macrophages).Examples of inflammation that result, at least in part, from a reactionof the non-specific defense system include inflammation associated withconditions such as adult (acute) respiratory distress syndrome (ARDS) ormultiple organ injury syndromes; reperfusion injury; acuteglomerulonephritis; reactive arthritis; dermatoses with acuteinflammatory components; acute purulent meningitis or other centralnervous system inflammatory disorders such as stroke; thermal injury;inflammatory bowel disease; granulocyte transfusion associatedsyndromes; and cytokine-induced toxicity.

[0187] “Autoimmune disease” as used herein refers to any group ofdisorders in which tissue injury is associated with humoral orcell-mediated responses to the body's own constituents. “Allergicdisease” as used herein refers to any symptoms, tissue damage, or lossof tissue function resulting from allergy. “Arthritic disease” as usedherein refers to any disease that is characterized by inflammatorylesions of the joints attributable to a variety of etiologies.“Dermatitis” as used herein refers to any of a large family of diseasesof the skin that are characterized by inflammation of the skinattributable to a variety of etiologies. “Transplant rejection” as usedherein refers to any immune reaction directed against grafted tissue(including organs or cells (e.g., bone marrow)), characterized by a lossof function of the grafted and surrounding tissues, pain, swelling,leukocytosis, and thrombocytopenia.

[0188] The therapeutic methods of the present invention include methodsfor the amelioration of disorders associated with inflammatory cellactivation. “Inflammatory cell activation” refers to the induction by astimulus (including, but not limited to, cytokines, antigens orauto-antibodies) of a proliferative cellular response, the production ofsoluble mediators (including but not limited to cytokines, oxygenradicals, enzymes, prostanoids, or vasoactive amines), or cell surfaceexpression of new or increased numbers of mediators (including, but notlimited to, major histocompatability antigens or cell adhesionmolecules) in inflammatory cells (including but not limited tomonocytes, macrophages, T lymphocytes, B lymphocytes, granulocytes(polymorphonuclear leukocytes including neutrophils, basophils, andeosinophils), mast cells, dendritic cells, Langerhans cells, andendothelial cells). It will be appreciated by persons skilled in the artthat the activation of one or a combination of these phenotypes in thesecells can contribute to the initiation, perpetuation, or exacerbation ofan inflammatory disorder.

[0189] The present invention enables methods of treating such diseasesas arthritic diseases, such as rheumatoid arthritis, osteoarthritis,gouty arthritis, spondylitis; Behcet disease; sepsis, septic shock,endotoxic shock, gram negative sepsis, gram positive sepsis, and toxicshock syndrome; multiple organ injury syndrome secondary to septicemia,trauma, or hemorrhage; ophthalmic disorders such as allergicconjunctivitis, vernal conjunctivitis, uveitis, and thyroid-associatedophthalmopathy; eosinophilic granuloma; pulmonary or respiratorydisorders such as asthma, chronic bronchitis, allergic rhinitis, ARDS,chronic pulmonary inflammatory disease (e.g., chronic obstructivepulmonary disease), silicosis, pulmonary sarcoidosis, pleurisy,alveolitis, vasculitis, pneumonia, bronchiectasis, and pulmonary oxygentoxicity; reperfusion injury of the myocardium, brain, or extremities;fibrosis such as cystic fibrosis; keloid formation or scar tissueformation; atherosclerosis; autoimmune diseases such as systemic lupuserythematosus (SLE), autoimmune thyroiditis, multiple sclerosis, someforms of diabetes, and Reynaud's syndrome; and transplant rejectiondisorders such as GVHD and allograft rejection; chronicglomerulonephritis; inflammatory bowel diseases such as Crohn's disease,ulcerative colitis and necrotizing enterocolitis; inflammatorydermatoses such as contact dermatitis, atopic dermatitis, psoriasis, orurticaria; fever and myalgias due to infection; central or peripheralnervous system inflammatory disorders such as meningitis, encephalitis,and brain or spinal cord injury due to minor trauma; Sjögren's syndrome;diseases involving leukocyte diapedesis; alcoholic hepatitis; bacterialpneumonia; antigen-antibody complex mediated diseases; hypovolemicshock; Type I diabetes mellitus; acute and delayed hypersensitivity;disease states due to leukocyte dyscrasia and metastasis; thermalinjury; granulocyte transfusion associated syndromes; andcytokine-induced toxicity.

[0190] The method has particular utility in treating humans who are ormay be subject to reperfusion injury, i.e., injury resulting fromsituations in which a tissue or organ experiences a period of ischemiafollowed by reperfusion. The term “ischemia” refers to localized tissueanemia due to obstruction of the inflow of arterial blood. Transientischemia followed by reperfusion characteristically results inneutrophil activation and transmigration through the endothelium of theblood vessels in the affected area. Accumulation of activatedneutrophils in turn results in generation of reactive oxygenmetabolites, which damage components of the involved tissue or organ.This phenomenon of “reperfusion injury” is commonly associated withconditions such as vascular stroke (including global and focalischemia), hemorrhagic shock, myocardial ischemia or infarction, organtransplantation, and cerebral vasospasm. To illustrate, reperfusioninjury occurs at the termination of cardiac bypass procedures or duringcardiac arrest when the heart, once prevented from receiving blood,begins to reperfuse. It is expected that inhibition of hPARP2 expressionor activity will result in reduced amounts of reperfusion injury in suchsituations.

[0191] With respect to the nervous system, global ischemia occurs whenblood flow to the entire brain ceases for a period. Global ischemia mayresult from cardiac arrest. Focal ischemia occurs when a portion of thebrain is deprived of its normal blood supply. Focal ischemia may resultfrom thromboembolytic occlusion of a cerebral vessel, traumatic headinjury, edema, or brain tumor. Even if transient, both global and focalischemia can cause widespread neuronal damage. Although nerve tissuedamage occurs over hours or even days following the onset of ischemia,some permanent nerve tissue damage may develop in the initial minutesfollowing the cessation of blood flow to the brain. Much of this damagehas been attributed to glutamate toxicity and to the secondaryconsequences of tissue reperfusion, such as the release of vasoactiveproducts by damaged endothelium and the release of cytotoxic products,such as free radicals and leukotrienes, by the damaged tissue.

[0192] Ischemia can also occur in the heart in myocardial infarction andother cardiovascular disorders in which the coronary arteries have beenobstructed as a result of atherosclerosis, thrombus, or spasm. Forexample, the method of the invention is believed to be useful fortreating cardiac tissue damage, particularly damage resulting fromcardiac ischemia or caused by reperfusion injury in mammals.

[0193] The invention further provides a method for treating aneurological disorder, comprising administering an neuroprotectiveamount of an hPARP2 antagonist. Neurological disorders that aretreatable by the method the present invention encompass disorders suchas peripheral neuropathy caused by physical injury or disease state;head trauma such as traumatic brain injury, physical damage to thespinal cord, stroke associated with brain damage, such as vascularstroke associated with hypoxia and brain damage, global or focalcerebral ischemia, and cerebral reperfusion injury; demyelinatingdiseases such as multiple sclerosis, and neurological disorders relatingto neurodegeneration. Examples of neurological disorders furtherinclude, without limitation, trigeminal neuralgia; glossopharyngealneuralgia; Bell's palsy; myasthenia gravis; muscular dystrophy;amyotrophic lateral sclerosis (ALS); progressive muscular atrophy;progressive bulbar inherited muscular atrophy; herniated, ruptured orprolapsed invertebrate disk syndromes; cervical spondylosis; plexusdisorders; thoracic outlet destruction syndromes; peripheralneuropathies such as those caused by lead, dapsone, ticks, porphyria, orGuillain-Barre syndrome; Alzheimer's disease; Huntington's Disease andParkinson's disease. The term “neurodegenerative diseases” includesAlzheimer's disease, Parkinson's disease, Huntington's disease, and ALS.

[0194] “Neuroprotective” as used herein refers to the effect ofreducing, arresting, or ameliorating nervous insult, or to the effect ofprotecting, resuscitating, or reviving nervous tissue that has sufferednervous insult.

[0195] “Nervous tissue” as used herein refers to the various componentsthat make up the nervous system including, without limitation, neurons,neural support cells, glia Schwann cells, vasculature contained withinand supplying these structures, the central nervous system, the brain,the brain stem, the spinal cord, the junction of the central nervoussystem with the peripheral nervous system, the peripheral nervoussystem, and allied structures.

[0196] As used herein, “nervous insult” refers to any damage to nervoustissue and any disability or death resulting therefrom. The cause ofnervous insult may be metabolic, toxic, neurotoxic, iatrogenic, thermalor chemical, and includes without limitation, vascular stroke, globaland focal ischemia, hypoxia, cerebrovascular accident, trauma, surgery,pressure, mass effect, hemorrhage, radiation, vasospasm,neurodegenerative disease, infection, Parkinson's disease, ALS,myelination/demyelination process, epilepsy, cognitive disorder,glutamate abnormality and secondary effects thereof.

[0197] “Preventing neurodegeneration” includes the ability to preventneurodegeneration in patients who have been diagnosed as having aneurodegenerative disease or who are at risk of developing aneurodegenerative disease. The term also encompasses preventing furtherneurodegeneration in patients who are already suffering from or havesymptoms of a neurodegenerative disease.

[0198] As used herein “nervous function” refers to the various functionsof the nervous system, which among other things provide an awareness ofthe internal and external environments of the body, make possiblevoluntary and reflex activities between the various. Structural elementsof the organism, and balance the organisms response to environmentalchanges.

[0199] Further, according to the invention, there is provided a methodof administering an hPARP2 antagonist to a human in an amount sufficientto effect a neuronal activity, particularly one that is not mediated byNMDA neurotoxicity. Such neuronal activity may consist of stimulation ofdamaged neurons, promotion of neuronal regeneration, prevention ofneurodegeneration and treatment of a neurological disorder.

[0200] The present invention also relates to a method of treating acardiovascular disorder in an animal, comprising administering to theanimal an effective amount of a compound that inhibits hPARP2 activity.“Cardiovascular disorders” as used herein refers to those disorders thatcan either cause ischemia or are caused by reperfusion of the heart.Examples include, but are not limited to, coronary artery disease,angina pectoris myocardial infarction, cardiovascular tissue damagecaused by cardiac arrest, cardiovascular tissue damage caused by cardiacbypass, cardiogenic shock, and related conditions that would be known bythose of ordinary skill in the art or that involve dysfunction of ortissue damage to the heart or vasculature, especially, but not limitedto, tissue damage related to PARP activation. The methods of theinvention are especially helpful in treating the acute forms of theabove cardiovascular disorders.

[0201] The invention also relates to a method of treating neoplastictissue growth, e.g., cancer, in an animal, comprising administering tothe animal an effective amount of a compound that inhibits hPARP2activity. In this embodiment, the method may further comprise adjuvantadministration of a chemotherapeutic or anti-cancer drug and/orradiation therapy.

[0202] Tumors or neoplasms include new growths of tissue in which themultiplication of cells is uncontrolled and progressive. Some suchgrowths are benign, but others are termed “malignant,” leading to deathof the organism. Malignant neoplasms or “cancers” are distinguished frombenign growths in that, in addition to exhibiting aggressive cellularproliferation, cancers invade surrounding tissues and metastasize.Moreover, malignant neoplasms are characterized in that they show agreater loss of differentiation (greater “dedifferentiation”), and oftheir organization relative to one another and their surroundingtissues. This property is also called “anaplasia.”

[0203] Neoplasms treatable by the present invention include solidtumors, i.e., carcinomas and sarcomas. Carcinomas include thosemalignant neoplasms derived from epithelial cells which tend toinfiltrate (invade) the surrounding tissues and give rise to metastases.Adenocarcinomas are carcinomas derived from glandular tissue or in whichthe tumor cells form recognizable glandular structures. Another broadcategory of cancers includes sarcomas, which are tumors whose cells areembedded in a fibrillar or homogeneous substance like embryonicconnective tissue. The invention also enables treatment of cancers ofthe myeloid or lymphoid systems, including leukemias, lymphomas andother cancers that typically do not present as a tumor mass, but aredistributed in the vascular or lymphoreticular systems.

[0204] The type of cancer or tumor cells amenable to treatment accordingto the invention include, for example, ACTH-producing tumor, acutelymphocytic leukemia, acute nonlymphocytic leukemia, cancer of theadrenal cortex, bladder cancer, brain cancer, breast cancer, cervicalcancer, chronic lymphocytic leukemia, chronic myelocytic leukemia,colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer,esophageal cancer, Ewing's sarcoma, gallbladder cancer, hairy cellleukemia, head and neck cancer, Hodgkin's lymphoma, Kaposi's sarcoma,kidney cancer, liver cancer, lung cancer (small and non-small cell),malignant peritoneal effusion, malignant pleural effusion, melanoma,mesothelioma, multiple myeloma, neuroblastoma, glioma, non-Hodgkin'slymphoma, osteosarcoma, ovarian cancer, ovarian (germ cell) cancer,pancreatic cancer, penile cancer, prostate cancer, retinoblastoma, skincancer, soft tissue sarcoma, squamous cell carcinomas, stomach cancer,testicular cancer, thyroid cancer, trophoblastic neoplasms, uterinecancer, vaginal cancer, cancer of the vulva, and Wilm's tumor.

[0205] The invention further relates to radiosensitizing tumor cells.The term “radiosensitizer” as used herein is defined as a molecule,preferably a low molecular weight molecule, administered to a human orother animal in therapeutically effective amounts to increase thesensitivity of the cells to electromagnetic radiation and/or to promotethe treatment of diseases that are treatable with electromagneticradiation. Diseases that are treatable with electromagnetic radiationinclude neoplastic diseases, benign and malignant tumors, and cancerouscells. Electromagnetic radiation treatment of other diseases not listedherein is also contemplated by the present invention. The term“radiation” as used herein includes, but is not limited to,electromagnetic radiation having wavelengths in the range of 10⁻²⁰ to10⁰ meters. Preferred embodiments of the present invention employgamma-radiation (10⁻²⁰ to 10⁻¹³ m), X-ray radiation (10⁻¹² to 10⁻⁹ m),ultraviolet light (10 nm to 400 nm), visible light (400 nm to 700 nm),infrared radiation (700 nm to 1.0 mm), or microwave radiation (1 mm to30 cm).

[0206] Radiosensitizers are known to increase the sensitivity ofcancerous cells to the toxic effects of electromagnetic radiation.Several mechanisms for the mode of action of radiosensitizers have beensuggested in the literature including: hypoxic cell radiosensitizers,e.g., 2-nitroimidazole compounds, and benzotriazine dioxide compounds)promote the reoxygenation of hypoxic tissue and/or catalyze thegeneration of damaging oxygen radicals; non-hypoxic cellradiosensitizers (e.g., halogenated pyrimidines) can be analogs of DNAbases and preferentially incorporate into the DNA of cancer cells andthereby promote the radiation ion-induced breaking of DNA moleculesand/or prevent the normal DNA repair mechanisms; and various otherpotential mechanisms of action have been hypothesized forradiosensitizers in the treatment of disease.

[0207] Many cancer treatment protocols currently employ radiosensitizersactivated by the electromagnetic radiation of X-rays. Examples of X-rayactivated radiosensitizers include, but are not limited to, thefollowing: metronidazole, misonidazole, desmethylmisonidazole,pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233,EO9, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR),5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine(FudR), hydroxyurea, cisplatin, and therapeutically effective analogsand derivatives thereof.

[0208] Photodynamic therapy (PDT) of cancers employs visible light asthe radiation activator of the sensitizing agent. Examples ofphotodynamic radiosensitizers include, but are not limited to:hematoporphyrin derivatives, Photofrin, benzoporphyrin derivatives,NPe6, tin etioporphyrin (SnET2), pheoborbide-a, bacteriochlorophyll-a,naphthalocyanines, phthalocyanines, zinc phthalocyanine, andtherapeutically effective analogs and derivatives thereof.

[0209] Radiosensitizers may be administered in conjunction with atherapeutically effective amount of one or more other compounds,including but not limited to: compounds that promote the incorporationof radiosensitizers to the target cells; compounds that control the flowof therapeutics, nutrients, and/or oxygen to the target cells;chemotherapeutic agents that act on the tumor with or without additionalradiation; or other therapeutically effective compounds for treatingcancer or other disease. Examples of additional therapeutic agents thatmay be used in conjunction with radiosensitizers include, but are notlimited to: 5-fluorouracil, leucovorin, 5′-amino-5′-deoxythymidineoxygen, carbogen, red cell transfusions, perfluorocarbons (e.g.,Fluosol-DA), 2,3-DPG, BW12C, calcium channel blockers, pentoxyfylline,anti-angiogenesis compounds, hydralazine, and L-BSO. Examples ofchemotherapeutic agents that may be used in conjunction withradiosensitizers include, but are not limited to: adriamycin,camptothecin, carboplatin, cisplatin, daunorubicin, docetaxel,doxorubicin, interferon (alpha, beta, gamma), interleukin 2, irinotecan,paclitaxel, topotecan, and therapeutically effective analogs andderivatives thereof.

[0210] The present invention further relates to use of hPARP2antagonists in methods of:

[0211] a) treating or preventing tissue damage resulting from celldamage or death due to necrosis or apoptosis, and conditions anddiseases related thereto including, but not limited to, renal failure,cachexia, retinal ischemia, skin aging, osteoarthritis, osteoporosis,chronic pain, acute pain, neuropathic pain, muscular dystrophy or otherdegenerative diseases of skeletal muscle involving replicativesenescence, age-related macular degeneration, AIDS and other immunesenescence diseases, and cancer;

[0212] b) extending the lifespan and proliferative capacity of cells;

[0213] c) altering gene expression in senescent cells by increasingexpression of young cell-specific genes and/or decreasing expression ofsenescent cell-specific genes and to extend or increase the lifespan orproliferative capacity of cells; and

[0214] d) treating disease or disease conditions induced or exacerbatedby cellular senescence such as skin aging.

[0215] The hparp2 polynucleotides provided by the invention also enabletherapeutic applications of these polynucleotides in treating thediseases and disorders described herein whose etiology involves hPARP2expression or activity. For example, an hparp2 antisense molecule mayprovide the basis for treatment of various abnormal conditions relatedto excessive or undesirable levels of poly(ADP-ribose) polymeraseactivity. Alternatively, polynucleotide sequences encoding hparp2 mayprovide the basis for the treatment of various abnormal conditionsrelated to deficiency of poly(ADP-ribose) polymerase activity.

[0216] Expression vectors derived from retroviruses, adenovirus, herpes,or vaccinia viruses, or from various bacterial plasmids, may be used fordelivery of recombinant hparp2 sense or antisense molecules to thetargeted cell population. Methods that are well known to those skilledin the art can be used to construct recombinant vectors containinghparp2 [see, e.g., Sambrook et al., supra, and Ausubel et al., supra].Alternatively, recombinant hparp2 can be delivered to target cells inliposomes.

[0217] The full-length cDNA sequence, and/or its regulatory elements,enables researchers to use an hparp2 polynucleotide as a tool in sense[Youssoufian and Lodish, Mol Cell Biol 13:98-104 (1993)] or antisense[Eguchi et al., Annu Rev Biochem 60:631-52 (1991)] investigations ofgene function. Oligonucleotides, designed from the cDNA or controlsequences obtained from the genomic DNA, can be used in vitro or in vivoto inhibit expression. Such technology is now well known in the art, andsense or antisense oligonucleotides or larger fragments can be designedfrom various locations along the coding or control regions.

[0218] Additionally, hPARP2 expression can be modulated by transfectinga cell or tissue with expression vectors that express high levels of anhparp2 polynucleotide fragment in conditions where it would bepreferably to block a biological activity of hPARP2. Such constructs canflood cells with untranslatable sense or antisense sequences. Even inthe absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until all copies of the vector are disabled byendogenous nucleases. Such transient expression may be accomplishedusing a non-replicating vector or a vector incorporating appropriatereplication elements.

[0219] Methods for introducing vectors into cells or tissue includethose methods discussed herein. In addition, several of thesetransformation or transfection methods are equally suitable for ex vivotherapy. Furthermore, the hparp2 polynucleotide sequences disclosedherein may be used in molecular biology techniques that have not yetbeen developed, provided the new techniques rely on properties ofnucleotide sequences that are currently known, including but not limitedto such properties as the triplet genetic code and specific base pairinteractions.

[0220] Pharmaceutical Compositions

[0221] The present invention further relates to pharmaceuticalcompositions that comprise a chemical or biological compound (“agent”)that is active as a modulator of hPARP2 expression or activity and abiocompatible pharmaceutical carrier, adjuvant, or vehicle. The activeagent in the pharmaceutical compositions may be selected from among allor portions of hparp2 polynucleotide sequences, hparp2 antisensemolecules, hPARP2 polypeptides, protein, peptide, or organic modulatorsof hPARP2 bioactivity, such as inhibitors, antagonists (includingantibodies) or agonists. Preferably, the agent is active in treating amedical condition that is mediated by or characterized by hPARP2expression or activity. The composition can include the agent as theonly active moiety or in combination with other nucleotide sequences,polypeptides, drugs, or hormones mixed with excipient(s) or otherpharmaceutically acceptable carriers.

[0222] Techniques for formulation and administration of pharmaceuticalcompositions may be found in Remington 's Pharmaceutical Sciences,18^(th) Ed., Mack Publishing Co, Easton Pa., 1990. The pharmaceuticalcompositions of the present invention may be manufactured using anyconventional method, e.g., mixing, dissolving, granulating,dragée-making, levigating, emulsifying, encapsulating, entrapping,melt-spinning, spray-drying, or lyophilizing processes. However, theoptimal pharmaceutical formulation will be determined by one of skill inthe art depending on the route of administration and the desired dosage.Such formulations may influence the physical state, stability, rate ofin vivo release, and rate of in vivo clearance of the administeredagent. Depending on the condition being treated, these pharmaceuticalcompositions may be formulated and administered systemically or locally.

[0223] The pharmaceutical compositions may be administered to thesubject by any conventional method, including parenteral and enteraltechniques. Parenteral administration modalities include those in whichthe composition is administered by a route other than through thegastrointestinal tract, for example, intravenous, intraarterial,intraperitoneal, intramedullary, intramuscular, intraarticular,intrathecal, and intraventricular injections. Enteral administrationmodalities include, for example, oral (including buccal and sublingual)and rectal administration. Transepithelial administration modalitiesinclude, for example, transmucosal administration and transdermaladministration. Transmucosal administration includes, for example,enteral administration as well as nasal, inhalation, and deep lungadministration; vaginal administration; and rectal administration.Transdermal administration includes passive or active transdermal ortranscutaneous modalities, including, for example, patches andiontophoresis devices, as well as topical application of pastes, salves,or ointments. Surgical techniques include implantation of depot(reservoir) compositions, osmotic pumps, and the like. A preferred routeof administration for treatment of inflammation would be local ortopical delivery for localized inflammation such as arthritis, andintravenous delivery for reperfusion injury or for systemic conditionssuch as septicemia.

[0224] The pharmaceutical compositions are formulated to containsuitable pharmaceutically acceptable carriers, and may optionallycomprise excipients and auxiliaries that facilitate processing of theactive compounds into preparations that can be used pharmaceutically.The administration modality will generally determine the nature of thecarrier. For example, formulations for parenteral administration maycomprise aqueous solutions of the active compounds in water-solubleform. Carriers suitable for parenteral administration can be selectedfrom among saline, buffered saline, dextrose, water, and otherphysiologically compatible solutions. Preferred carriers for parenteraladministration are physiologically compatible buffers such as Hank'ssolution, Ringer's solutions, or physiologically buffered saline. Fortissue or cellular administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art. For preparations comprisingproteins, the formulation may include stabilizing materials, such aspolyols (e.g., sucrose) and/or surfactants (e.g., nonionic surfactants),and the like.

[0225] Alternatively, formulations for parenteral use may comprisesuspensions of the active compounds prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils, such as sesame oil, and synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Aqueous injectionsuspensions may contain substances that increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions. Emulsions,e.g., oil-in-water and water-in-oil dispersions, can also be used,optionally stabilized by an emulsifying agent or dispersant(surface-active materials; surfactants). Liposomes containing the activeagent may also be employed for parenteral administration.

[0226] Alternatively, the pharmaceutical compositions comprising theagent in dosages suitable for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art. Thepreparations formulated for oral administration may be in the form oftablets, pills, capsules, cachets, dragées, lozenges, liquids, gels,syrups, slurries, suspensions, or powders. To illustrate, pharmaceuticalpreparations for oral use can be obtained by combining the activecompounds with a solid excipient, optionally grinding the resultingmixture, and processing the mixture of granules, after adding suitableauxiliaries if desired, to obtain tablets or dragée cores. Note thatoral formulations may employ liquid carriers similar in type to thosedescribed for parenteral use, e.g., buffered aqueous solutions,suspensions, and the like.

[0227] Preferred oral formulations include tablets, dragées, and gelatincapsules. These preparations may contain one or excipients, whichinclude, without limitation:

[0228] a) diluents such as sugars, including lactose, dextrose, sucrose,mannitol, or sorbitol;

[0229] b) binders such as magnesium aluminum silicate, starch from corn,wheat, rice, potato, etc.;

[0230] c) cellulose materials such as methyl cellulose,hydroxypropylmethyl cellulose, and sodium carboxymethyl cellulose,polyvinyl pyrrolidone, gums such as gum arabic and gum tragacanth, andproteins such as gelatin and collagen;

[0231] d) disintegrating or solubilizing agents such as cross-linkedpolyvinyl pyrrolidone, starches, agar, alginic acid or a salt thereofsuch as sodium alginate, or effervescent compositions;

[0232] e) lubricants such as silica, talc, stearic acid or its magnesiumor calcium salt, and polyethylene glycol;

[0233] f) flavorants, and sweeteners;

[0234] g) colorants or pigments, e.g., to identify the product or tocharacterize the quantity (dosage) of active compound; and

[0235] h) other ingredients such as preservatives, stabilizers, swellingagents, emulsifying agents, solution promoters, salts for regulatingosmotic pressure, and buffers.

[0236] Gelatin capsules include push-fit capsules made of gelatin, aswell as soft, sealed capsules made of gelatin and a coating such asglycerol or sorbitol. Push-fit capsules can contain the activeingredient(s) mixed with fillers, binders, lubricants, and/orstabilizers, etc. In soft capsules, the active compounds may bedissolved or suspended in suitable fluids, such as fatty oils, liquidparaffin, or liquid polyethylene glycol with or without stabilizers.

[0237] Dragée cores can be provided with suitable coatings such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures.

[0238] The pharmaceutical composition may be provided as a salt of theactive agent, which can be formed with many acids, including but notlimited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic,succinic, etc. Salts tend to be more soluble in aqueous or otherprotonic solvents that are the corresponding free base forms.

[0239] To be effective therapeutically in modulating central nervoussystem targets, the agents used in the methods of the invention shouldreadily penetrate the blood brain barrier when peripherallyadministered. Compounds that cannot penetrate the blood brain barrier,however, can still be effectively administered by an intravenous route.

[0240] As noted above, the characteristics of the agent itself and theformulation of the agent can influence the physical state, stability,rate of in vivo release, and rate of in vivo clearance of theadministered agent. Such pharmacokinetic and pharmacodynamic informationcan be collected through pre-clinical in vitro and in vivo studies,later confirmed in humans during the course of clinical trials. Thus,for any compound used in the method of the invention, a therapeuticallyeffective dose can be estimated initially from biochemical and/orcell-based assays. Then, dosage can be formulated in animal models toachieve a desirable circulating concentration range that modulateshPARP2 expression or activity. As human studies are conducted, furtherinformation will emerge regarding the appropriate dosage levels andduration of treatment for various diseases and conditions.

[0241] Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the “therapeutic index,” which is typically expressed as theratio LD₅₀/ED₅₀. Compounds that exhibit large therapeutic indices arepreferred. The data obtained from such cell culture assays andadditional animal studies can be used in formulating a range of dosagefor human use. The dosage of such compounds lies preferably within arange of circulating concentrations that include the ED₅₀ with little orno toxicity.

[0242] For the method of the invention, any effective administrationregimen regulating the timing and sequence of doses may be used. Dosesof the agent preferably include pharmaceutical dosage units comprisingan effective amount of the agent. As used herein, “effective amount”refers to an amount sufficient to modulate hPARP2 expression or activityand/or derive a measurable change in a physiological parameter of thesubject through administration of one or more of the pharmaceuticaldosage units.

[0243] Exemplary dosage levels for a human subject are of the order offrom about 0.001 milligram of active agent per kilogram body weight(mg/kg) to about 100 mg/kg. Typically, dosage units of the active agentcomprise from about 0.01 mg to about 10,000 mg, preferably from about0.1 mg to about 1,000 mg, depending upon the indication, route ofadministration, etc. Depending on the route of administration, asuitable dose may be calculated according to body weight, body surfacearea, or organ size. The final dosage regimen will be determined by theattending, physician in view of good medical practice, consideringvarious factors that modify the action of drugs, e.g., the agent'sspecific activity, the severity of the disease state, the responsivenessof the patient, the age, condition, body weight, sex, and diet of thepatient, the severity of any infection, etc. Additional factors that maybe taken into account include time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy. Further refinement of the dosage appropriate for treatmentinvolving any of the formulations mentioned herein is done routinely bythe skilled practitioner without undue experimentation, especially inlight of the dosage information and assays disclosed, as well as thepharmacokinetic data observed in human clinical trials. Appropriatedosages may be ascertained through use of established assays fordetermining concentration of the agent in a body fluid or other sampletogether with dose response data.

[0244] The frequency of dosing will depend on the pharmacokineticparameters of the agent and the route of administration. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Accordingly, thepharmaceutical compositions can be administered in a single dose,multiple discrete doses, continuous infusion, sustained release depots,or combinations thereof, as required to maintain desired minimum levelof the agent. Short-acting pharmaceutical compositions (i.e., shorthalf-life) can be administered once a day or more than once a day (e.g.,two, three, or four times a day). Long acting pharmaceuticalcompositions might be administered every 3 to 4 days, every week, oronce every two weeks. Pumps, such as subcutaneous, intraperitoneal, orsubdural pumps, may be preferred for continuous infusion.

[0245] Compositions comprising a compound of the invention formulated ina pharmaceutical acceptable carrier may be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition. Conditions indicated on the label may include treatment ofinflammatory disorders, cancer, nervous tissue injury, etc. Kits arealso contemplated, wherein the kit comprises a dosage form of apharmaceutical composition and a package insert containing instructionsfor use of the composition in treatment of a medical condition.

[0246] The following Examples are provided to further aid inunderstanding the invention. The particular materials and conditionsemployed are intended to exemplify particular aspects of the inventionand should not be construed to limit the reasonable scope thereof.

[0247] The Examples presuppose an understanding of conventional methodswell-known to those persons having ordinary skill in the art to whichthe examples pertain, e.g., the construction of vectors and plasmids,the insertion of genes encoding polypeptides into such vectors andplasmids, or the introduction of vectors and plasmids into host cells.Such methods are described in detail in numerous publications including,for example, Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press (1989); Ausubel et al. (Eds.),Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994);and Ausubel et al. (Eds.), Short Protocols in Molecular Biology, 4^(th)ed., John Wiley & Sons, Inc. (1999).

EXAMPLE 1

[0248] Identification of a Human EST Related to Mouse parp2

[0249] Using the nucleotide sequence of mouse parp2 [National Center forBiotechnology Information (NCBI) GenBank® Accession number AJ007780 (SEQID NO:22)], a search of NCBI Expressed Sequence Tags (EST) database wasperformed to investigate the possibility that a human homologue of themouse PARP2 gene might exist. The EST database provides 5′ and/or 3′nucleotide sequences for cDNA clones from a variety of tissue sources.The NCBI BLASTn (Basic Local Alignment Search Tool—nucleotide) programwas used to compare the nucleotide query sequence of mouse PARP2 againsta nucleotide sequence database and to identify DNA sequences in thehuman EST sequence database that have significant homology to mousePARP2. This BLASTn search identified an EST sequence designated AA568817(SEQ ID NO:3), cloned from human colon. Regions of homology of the ESTto the mouse parp2 nucleic acid sequence were identified. Specifically,nucleotides 119 to 284 (nt 119-284) of AA568817 shared substantialhomology with the antisense complement nt 1536-1701 of mouse parp2,being identical at 150 of 166 nucleotides (90% identity). Moreover, theamino acid sequence predicted from nt 128-283 of the antisense strand ofAA568817 corresponded to a region consisting of amino acids 508 to 559(aa 508-559) of the mouse PARP2 protein (SEQ ID NO:23), wherein theproteins were identical at 48 of the 52 amino acid positions (92%identity).

[0250] AA568817 was used in a search of the GenBank® database using theNCBI UniGene® program in order to identify other human EST sequencesoriginating from the same gene. The UniGene® program assembles GenBank®sequences into a non-redundant set of gene-oriented clusters, with eachcluster containing a group of sequences from the same gene. The UniGene®search of the human GenBank® database with AA568817 identifiedthirty-four human EST sequences belonging in the same gene cluster asAA568817. One of these human ESTs, designated R28358, (SEQ ID NO:4), wascloned from human placenta and contained 3′ sequence from an I.M.A.G.E(Integrated Molecular Analysis of Genome Expression Consortium) clone,designated 133650. I.M.A.G.E is a consortium coordinated by LawrenceLivermore National Laboratory (Livermore, CA) that sequences cDNAs andmakes them publicly available through the American Type CultureCollection (ATCC; Rockville, Md.). R28358 was used in a search of theATCC database to identify EST R28562 (SEQ ID NO:5) as the 5′ sequencedend of I.M.A.G.E clone 133650.

[0251] R28358 was compared to the antisense sequence of mouse parp2. Itwas found that nt 110-240 of R28358 shared significant homology with nt1563-1692 of mouse parp2 (108 of 128 nucleotides were identical; 84%identity). The antisense strand of R28358 (nt 110-160) was translatedand the predicted protein was compared with mouse PARP2 protein (nt1642-1692 translated to aa 543-559). This comparison revealed that theproteins were the same at 16 of the 17 corresponding amino acidpositions (94% identity).

[0252] R28562 was compared to mouse parp2. Nucleotides 4-186 of R28562were found to share homology with nt 952-1134 of mouse parp2 (159 of 183nucleotides were identical; 84% identity). When nt 4-186 of R28562 weretranslated and the predicted protein compared with the correspondingregion of mouse PARP2 protein (nt 952-1134 translated to aa 313-373),the proteins were found to be the same at 54 of 61 corresponding aminoacid positions (89% identity).

EXAMPLE 2

[0253] Isolation of Full-Length hPARP2-Encoding Polynucleotide

[0254] To clone the 5′ end of human hparp2, 5′ RACE analysis wasperformed using human Marathon™-Ready spleen and testis cDNA libraries(Clontech) as the templates. A primer corresponding to the antisensestrand of EST R28562 (SEQ ID NO:6) was synthesized for use in apolymerase chain reaction with the API primer (Clontech; SEQ ID NO:7)that was designed to anneal to the Marathon™ cDNA Adapters ligated tothe ends of the cDNA libraries.

[0255] R28562 Antisense R28562 Antisense (SEQ ID NO:6)GTGTTGGTCCAATGGGTGTTCTGGGCTTTGTAGCTCTG AP1 (SEQ ID NO:7)CCATCCTAATACGACTCACTATAGGGC

[0256] The PCR reaction contained 5 μL human spleen or testisMarathon™-Ready cDNA, 0.20 μM each primer, 0.20 mM dNTPs, 1×PCR buffer,and 1 μL of Advantage® cDNA Taq Polymerase mix (Clontech). The reactionwas performed in a GeneAmp® PCR System 9700 machine (PE AppliedBiosystems, Norwalk, Conn.) with the following four steps: 1) 1 cycle at94° C. for 1 min; 2) 5 cycles of 94° C. for 30 sec and 72° C. for 4 min;3) 5 cycles of 94° C. for 30 sec and 70° C. for 4 min; and 4) 25 cyclesof 94° C. for 30 sec, and 60° C. for 4 min. The PCR fragment, designated5′-hPARP2, was isolated using gel electrophoresis and a QIAquick® GelExtraction Kit (QIAGEN, Valencia, Calif.) according to themanufacturer's instructions.

[0257] 5′-hPARP2 was cloned directly into pCR®2.1-TOPO vector(Invitrogen, Carlsbad, Calif.), according to the manufacturer'sinstructions. Because Taq polymerase has an error rate of 8.0×10⁻⁶mutation/base pair [Cline et al., Nucleic Acids Res 24(18):3546-51(1996)], four unique clones were sequenced and compared to eliminate thepossibility of Taq polymerase-induced errors in the sequence of5′-hPARP2. The four clones were determined to be unique by theirdifferent lengths. The differences in the lengths of the four clonesindicated that they were amplified from unique clones in the Marathon™libraries.

[0258] The four unique clones of 5′-hPARP2 were sequenced with primersthat hybridized to the vector DNA: M13 Forward TGTAAAACGACGGCCAGT (SEQID NO:8) M13 Reverse GGAAACAGCTATGACCATG (SEQ ID NO:9)

[0259] and primers designed to anneal to the cDNA sequence: (SEQ IDNO:10) 3-P2-SEQ1 GGCTGTACACTCTGGGTCCACAGGAGC (SEQ ID NO:11) 3-P2-SEQ2CTTCCATGAGAGCTCGTCCATGCTGGCC (SEQ ID NO:12) 5-P2-SEQ2GGCCAGCATGGACGAGCTCTCATGGAAG.

[0260] The four individual nucleotide sequences were compiled into aconsensus nucleotide sequence designated 5′-hPARP2 (SEQ ID NO:13). Inthe consensus nucleotide sequence of 5′-hPARP2, every base pair waspresent at the corresponding position in three of the four uniqueclones, except consensus sequence nucleotides 625, 632, and 881., whichwere present in two of the four unique clones.

[0261] To confirm that nucleotides 625, 632, and 881 of the 5′-hPARP2consensus sequence were correct, two separate PCR reactions wereperformed using the Marathon™-Ready human testis cDNA library (Clontech)as the template. A primer corresponding to the sense strand of 5′-hPARP2(5-P2-SEQ1; SEQ ID NO:14) and primers corresponding to the antisensestrand of EST R28358 (designated hPARP2 Li (SEQ ID NO:15) and hPARP2 L2(SEQ ID NO:16)) were used in PCR reactions under the conditionsdescribed previously. 5-P2-SEQ1 GCTCCTGTGGACCCAGAGTGTACAGCC (SEQ IDNO:14) hPARP2 L1 ACATTCACCACAGCTGAAGG (SEQ ID NO:15) hPARP2 L2CCACAGCTGAAGGAAATTAAAC (SEQ ID NO:16)

[0262] The PCR fragments, designated P2-1 (amplified with 5-P2-SEQ1 andhPARP2 L1) and P2-9 (amplified with 5-P2-SEQ1 and hPARP2 L2), wereisolated using gel electrophoresis and a QIAquick® Gel Extraction Kit(QIAGEN) according to the manufacturer's instructions.

[0263] P2-1 and P2-9 were cloned directly into the pCR®2.1-TOPO vector(Invitrogen) according to the manufacturer's instructions. P2-1 and P2-9were sequenced with the M13 Forward and M13 Reverse primers thathybridize to the vector DNA (SEQ ID NOs:8 and 9, respectively) and thepartial nucleotide sequences of P2-1 and P2-9 are set out in SEQ IDNO:17 and SEQ ID NO:18, respectively. Nucleotides 625, 632, and 881 ofthe 5′-hPARP2 consensus sequence were determined to be present in clonesP2-1 and P2-9 (nucleotides 267, 274, and 523, respectively), and thus,determined to be correct. 5′-hPARP2 was determined to have an openreading frame (ORF) of 1080 nucleotides beginning at nucleotide 63. Thededuced amino acid sequence from nucleotides 63-1142 of 5′-hPARP2 is setout in SEQ ID NO:19.

[0264] To clone the 3′ end of human hparp2, two separate PCR reactionswere performed using the Marathon™-Ready human testis cDNA library(Clontech) as the template. A primer corresponding to the sense strandof 5′-hPARP2 (5-P2-SEQ2; SEQ ID NO:14) and primers corresponding to theantisense strand of EST R28358 (hPARP2 L1, SEQ ID NO:15; hPARP2 L2, SEQID NO:16) were used in PCR reactions under the conditions describedpreviously. The PCR fragments, designated 3′-hPARP2-L1 (amplified with5-P2-SEQ2 and hPARP2 LI) and 3′-hPARP2-L2 (amplified with 5-P2-SEQ2 andhPARP2 L2), were isolated using gel electrophoresis and a QIAquick® GelExtraction Kit (QIAGEN) according to the manufacturer's instructions.

[0265] 3′-hPARP2-L1 and 3′-hPARP2-L2 were cloned directly into thepCR®2.1-TOPO vector (Invitrogen) according to the manufacturer'sinstructions. Four clones of 3′-hPARP2-L1 and three clones of3′-hPARP2-L2 were sequenced with primers that hybridized to the vectorDNA (SEQ ID NO:8 and SEQ ID NO:9) to eliminate the possibility of Taqpolymerase-induced errors in the sequence of 3′-hPARP2 as discussedabove. The seven nucleotide sequences were compiled into a consensusnucleotide sequence, designated 3′-hPARP2, which is set out in SEQ IDNO:20.

[0266] Every base pair in the consensus nucleotide sequence of 3′-hPARP2was present at the corresponding position in at least six of the sevenclones used to compile the consensus, except nt 856-864, which werepresent in at least three of the 7 clones. However, the consensussequence of nt 856-864 of 3′-hPARP2 was present in EST R28358, and thus,was determined to be correct. Nucleotides 1-193 of 3′-hPARP2 weredetermined to overlap with 5′-hPARP2 (nt 951-1143). 3′-hPARP2 had 671additional nucleotides located 3′ to the overlapping region (nt194-864). 3′-hPARP2 was determined to have an ORF of 861 nucleotidesbeginning at nt 1, with a stop codon beginning at nt 862. The amino acidsequence deduced from nt 1-861 of 3′-hPARP2 is set out in SEQ ID NO:21.

[0267] 5′-hPARP2 (nt 1-1143) was joined with 3′-hPARP2(nt 194-864), andthe resultant polynucleotide sequence was designated “hparp2” (humanparp2) and is set out in SEQ ID NO:1. A comparison of the hparp2 andmouse parp2 (SEQ ID NO:22) sequences revealed that nt 306-728 of hparp2shared substantial homology with nt 199-621 of mouse parp2 (374 of 423nucleotides were identical; 88% identity). In addition, nt 744-1814 ofhparp2 shared substantial homology with nt 625-1695 of mouse parp2 (943of 1071 nucleotides were identical; 88% identity).

[0268] hparp2 was determined to have an ORF of 1789 nucleotidesbeginning at nt 63 and to have a stop codon beginning at nt 1812. TheATG beginning at nt 63 was determined to be the initiating methioninecodon by the presence of upstream in frame stop codons. The deducedamino acid sequence from nt 63-1811 of hparp2 is set out in SEQ ID NO:2.hPARP2 and mouse PARP2 (SEQ ID NO:23) were the same at 489 of 558 aminoacid residues (88% identity). Twenty-six amino acid residue gaps in thealignment were present: hPARP2 amino acid residues 8, 14, 45-48, 58,68-81, and 223-226 did not align with mouse PARP2; and mouse PARP2residue 34 did not align with hPARP2.

[0269] The human parp1 gene (SEQ ID NO:24) encodes a protein hPARP1 (SEQID NO:25) containing a 46 kDa amino-terminal DNA binding domain (aa1-373 of SEQ ID NO:25) that includes two zinc-finger motifs and anuclear localization signal [see Molinete et al., “Structure andfunction of the human poly(ADP-ribose) polymerase,” ADP-RibosylationReactions, Poirier and Moreau (Eds.), Springer-Verlag, New York, 1992,at pp. 3-13]. hPARP2 does not contain an obvious zinc-finger DNA bindingdomain, but it may contain a different, yet unidentified, DNA bindingdomain. hPARP2 may also interact with DNA indirectly by binding to a DNAbinding protein. It is also possible that hPARP2 does not interactdirectly or indirectly with DNA.

[0270] hPARP1 contains a central 22 kDa automodification domain (aa373-525 of SEQ ID NO:25) that contains 15 glutamic acid residue sites,of which some or all may be sites for automodification [Pieper et al.,Trends Pharmacol Sci 20:171-81 (1999)]. The automodification domain ofhPARP1 does not align with hPARP2, but hPARP2 does contain 44 glutamicacid residues that may be sites for automodification.

[0271] HPARP1 contains a carboxyl terminal 40 kDa catalytic domain (aa655-1014 of SEQ ID NO:25) [Molinete et al., supra]. When aa 224-583 ofhPARP2 (SEQ ID NO:2) are aligned with aa 655-1014 of hPARP1, 145 of 364amino acids are the same (40% identity). A “G-X-X-X-G-K-G” motifinvolved in catalytic activity also is conserved between hPARP1 (aa888-894 of SEQ ID NO:25) and hPARP2 (aa 454-460 of SEQ ID NO:2).

EXAMPLE 3

[0272] Measurement of hPARP2 Biological Activity

[0273] PARP1 has endogenous poly(ADP) polymerase activity that isactivated when PARP1 is bound to damaged DNA. This activation can bereadily assayed due to the presence of an automodification domain inhPARP1 [see, e.g., Nishikimi et al., J Biol Chem 257:6102-5 (1982)]. Thestructural similarity of hPARP2 to hPARP1 suggests that hPARP2 will alsopossess poly(ADP) polymerase activity. However, structural differencesin the amino terminal half of the molecules make it uncertain whetherhPARP2 will be 1) activated by DNA damage, or 2) a target ofautomodification.

[0274] The activation of hPARP2 by DNA can be readily assessed byemploying methods developed for the measurement of polymerase activityby PARP1 in the presence or absence of DNA [see, e.g., Benjamin andGill, J Biol Chem 255:10502-8 (1980); Yoshihara, Biochem Biophys ResCommun 47:119-25 (1972)]. DNA samples may be prepared to contain singlestranded or double stranded DNA, closed circular DNA, or linear DNA,with blunt, 3′-recessed, or 5′-recessed ends. The automodification ofhPARP2 can readily be determined by measurement of incorporation ofADP-ribose into forms covalently attached to the hPARP2 protein usingmethods developed for PARP1 [e.g., Banasik et al., supra].

[0275] Alternatively, one method for the determination of hPARP2catalytic activity, in the absence of information regarding DNA bindingactivity or automodification activity, is to substitute the catalyticregion of hPARP2 for the catalytic region of hPARP1 in the hPARP1 cDNA.This can be accomplished using the standard tools of molecular biology.The expression and purification of such a chimeric protein allows theassessment of hPARP2 catalytic activity in a context that is independentof hPARP2-specific activation or substrates. In this case, theDNA-binding domain of hPARP1 may permit DNA-dependent activation of thehPARP2 catalytic domain. Likewise, the automodification domain of hPARP1may serve as a target of hPARP2-mediated ADP-ribosylation.

[0276] If the ability of hPARP2 to automodify either in the form ofnative polypeptide or chimeric polypeptide is undetectable or difficultto detect, an alternative approach is to add to the assay heterologousproteins that might be expected to serve as a target of ADP-ribosylation[e.g., Tsopanakis et al., Eur J Biochem 90:337-45 (1978)].

[0277] Further characterization of biological activities of hPARP2 canalso be obtained by ectopic expression of the hPARP2 polypeptide.Ectopic expression can be induced, for example, by transfection ofhPARP2 expression vectors into cultured cell lines. Analysis of thephenotype of transiently or stably transfected cell lines can be usedfor the determination of biological function. Such experiments arecommon in the art [see, e.g., Fritz et al., Mutation Res 308:127-33(1994)]. Conversely, targeted disruption of hPARP2 expression oractivity by, for example, the use of gene activation. antisenseoligonucleotides., or hPARP2-specific chemical inhibitors. can be usedfor the identification of hPARP2-specific biological function.

[0278] Construction of Baculovirus Expression Plasmids

[0279] The primary structure of the hparp2 polypeptide suggests thathPARP2, like hPARP1, will have poly(ADP-ribose) polymerase activity. ThePARP activity of hPARP2, or some substructure thereof, can be measuredby the ability of that component to incorporate the ADP-ribose unit fromNAD into polymers of ADP-ribose coupled to a protein substrate. Thedemonstration of such activity on a given substrate is readilyaccomplished by the skilled artisan [see, for example, Smith et al.,Science 282:1484-1487 (1998)].

[0280] A fusion protein, designated PARP1A/PARP2B, containing aa 1-662of hPARP1 (SEQ ID NO:25) fused upstream of aa 230-583 of hPARP2 (SEQ IDNO:2) was used in the measurement of hPARP2 poly(ADP-ribose) polymeraseactivity. PARP1A/PARP2B contained the DNA binding domain (aa 1-373 ofSEQ ID NO:25) and automodification domain (aa 373-525 of SEQ ID NO:25)of hPARP1 and the putative catalytic domain of hPARP2 (aa 224-583 of SEQID NO:2).

[0281] The PARP1A piece of the fusion protein was amplified by PCR usinga primer (Sal-PARP1; SEQ ID NO:26) corresponding to the sense strand ofhparp1 polynucleotide sequence (nt 1-30 of SEQ ID NO:24) and a primer(revMlu-PARP1; SEQ ID NO:27) corresponding to the antisense strand ofhparp1 polynucleotide sequence (nt 1957-1985 of SEQ ID NO:24). Sal-PARP1(SEQ ID NO:26) CGTCGACCCATGGCGGAGTCTTCGGATAAGCTCTATCGA revMlu-PARP1 (SEQID NO:27) GGAAACGCGTTTGGTGCCAGGATTTACTGTCAGCTTCTT

[0282] The PCR reaction contained 0.5 μL of human thymus or testisQUICK-Clone™ cDNA (Clontech), 0.25 μM each primer, 0.20 mM dNTPs, 1×PCRbuffer, and 1 μL of Clontech Advantage® polymerase mix. The reactionswere performed in a GeneAmp® PCR System 9700 machine (PE AppliedBiosystems) with the following steps: 1) 1 cycle at 94° C. for 1 min; 2)30 cycles of 94° C. for 30 sec, 60° C. for 2 min, and 72° C. for 2 min;and 3) 1 cycle at 72° C. for 7 min. The PCR fragment (designatedhparp1A) was isolated using gel electrophoresis and a QIAquick® GelExtraction Kit (QIAGEN) according to the manufacturer's instructions.hparp1A was subcloned into the pTrcHis2™-Topo™ vector (Invitrogen)according to the manufacturer's instructions. hparp1A was digested frompTrcHis2™-Topo™ with SalI and MluI, the fragment isolated using gelelectrophoresis and a QIAquick® Gel Extraction Kit (QIAGEN), and savedfor further subcloning described below.

[0283] The PARP2B piece of the fusion protein was amplified by PCR usinga primer (for Mlu-PARP2; SEQ ID NO:28) corresponding to the sense strandof hparp2 polynucleotide sequence (nt 750-776 of SEQ ID NO:1) and aprimer (PARP2-Strep-Not; SEQ ID NO:29) corresponding to the antisensestrand of hparp2 polynucleotide sequence (nt 1771-1811 of SEQ ID NO:1).forMlu-PARP2 (SEQ ID NO:28) TTGAAACGCGTTCCAGAGTCACAGCTAGATCTTCGGGTAPARP2-Strep-Not (SEQ ID NO:29)GTCTCGAAAGCGGCCGCTTAGCCTCCGAACTGTGGATGCCTCCACGCCCACAGCTGAAGGAAATTAAACTGAACCTTTAAAAGGTACC

[0284] The PCR reaction contained 100 ng hparp2 cDNA, 0.25 μM eachprimer, 0.20 mM dNTPs, 1×PCR buffer, and 1 μL of Clontech Advantage®polymerase mix. The reactions were performed in a GeneAmp® PCR System9700 machine (PE Applied Biosystems) with the following steps: 1) 1cycle at 94° C. for 1 min; 2) 30 cycles of 94° C. for 30 sec, 60° C. for2 min, and 72° C. for 2 min; and 3) 1 cycle at 72° C. for 7 min. The PCRfragment (designated hparp2B) was isolated using gel electrophoresis anda QIAquick® Gel Extraction Kit (QIAGEN) according to the manufacturer'sinstructions. The hparp2B fragment was cloned into thepcDNA3.1I/NT-GFP-TOPO™ vector (Invitrogen) according to themanufacturer's instructions.

[0285] hparp2B was digested from pcDNA3.1/NT-GFP-TOPO™ with MluI andNotI and subcloned with SalI/MluI digested hparp1A (see above) into apFASTBAC vector (Gibco BRL, Rockville, Md.) that had previously beendigested with SalI and NotI. The resultant plasmid was designatedpFB-PARP1A/PARP2B.

[0286] pFB-PARP1A/PARP2B was sequenced with primers designed to annealto the vector sequence (SEQ ID NOs:30 and 31) and primers designed toanneal to the cDNA sequence (SEQ ID NOs:6, 11, and 32-45).Vector primers for PARP1A/PARP2B (SEQ ID NO:30) FastBac forTTTGTTCGCCCAGACTC (SEQ ID NO:31) FastBac rev TATGTTTCAGGTTCAGGGGGAGcDNA Primers for PARP1A/PARP2B (SEQ ID NO:32) P1 GCGGAAGCTGGAGGAGTGAC(SEQ ID NO:33) P2 GTCACTCCTCCAGCTTCCGC (SEQ ID NO:34) P3AAGCCCTGAAGAAGCAGCTC (SEQ ID NO:35) P4 GAGCTGCTTCTTCAGGGCTT (SEQ IDNO:36) P5 CAGACACCCAACCGGAAGGA (SEQ ID NO:37) P6 TCCTTCCGGTTGGGTGTCTG(SEQ ID NO:38) P7 TCCGCCTCCACCAAGAGCCT (SEQ ID NO:39) P8AGGCTCTTGGTGGAGGCGGA (SEQ ID NO:40) P9 TGGCCTGGTGGACATCGTTA (SEQ IDNO:41) P10 TAACGATGTCCACCAGGCCA (SEQ ID NO:11) 3-P2-SEQ2CTTCCATGAGAGCTCGTCCATGCTGGCC (SEQ ID NO:6) 3-PARP2GTGTTGGTCCAATGGGTGTTCTGGGCTTTGTAGCTCTG (SEQ ID NO:42) AA#5GTATTCTTTAGGCGAGAGGC (SEQ ID NO:43) H23985-5 TGACGAAGTGGGCAGAACTG (SEQID NO:44) PARP2U2 REV GAGCACCCCCTGGACCAGCAC (SEQ ID NO:45) AA#3ACAGCGACTATACCATGACC

[0287] The nucleotide sequence of PARP1A/PARP2B is set out in SEQ IDNO:46, and the amino acid sequence of PARP1A/PARP2B is set out in SEQ IDNO:47. PARP1A/PARP2B consists of the following regions: a His tag leaderregion at aa 1-36; a hPARP1 region at aa 37-698; a spacer region fromamino acids 699 to 700; a hPARP2 region at aa 701-1054; and a Strep-tagregion at aa 1055-1063.

[0288] In addition to PARP1A/PARP2B, a full-length hPARP2 protein fusedto a poly-His tag (FB-hPARP2) was constructed for use in the measurementof hPARP2 poly(ADP-ribose) polymerase activity. The construction ofFB-hPARP2 was carried out as follows.

[0289] A carboxyl region of hparp2 was isolated by digesting thepFB-PARP1A/PARP2B plasmid with SalI and SacI to remove the hPARP1region. This hPARP2 region (pFB-PARP2B-Sal/Sac) was isolated using gelelectrophoresis and a QIAquick® Gel Extraction Kit (QIAGEN) and savedfor further subcloning described below.

[0290] An amino terminal region of hPARP2 was amplified by PCR using aprimer (Sal-PARP2; SEQ ID NO:48) corresponding to the sense strand ofhparp2 polynucleotide sequence (nt 63-92 of SEQ ID NO:1) and a primer(revMlu-PARP2; SEQ ID NO:49) corresponding to the antisense strand ofhparp2 polynucleotide sequence (nt 720-748 of SEQ ID NO:1). Sal-PARP2(SEQ ID NO:48) CGTCGACCCATGGCGGCGCGGCGGCGACGGAGCACCGGC revMlu-PARP2 (SEQID NO:49) TGGAACGCGTTTCAAGGGAGATTTAAGAGATTCCTCTTT

[0291] The PCR reaction contained 100 ng of hparp2 cDNA, 0.25 μM eachprimer, 0.20 mM dNTPs, 1×PCR buffer, and 1 μL of Clontech Advantage®polymerase mix. The reactions were performed in a GeneAmp® PCR System9700 machine (PE Applied Biosystems) with the following steps: 1) 1cycle at 94° C. for 1 min; 2) 30 cycles of 94° C. for 30 sec, 60° C. for2 min, and 72° C. for 2 min; and 3) 1 cycle at 72° C. for 7 min. The PCRfragment (designated hparp2-Sal/Mlu) was isolated using gelelectrophoresis and a QIAquick® Gel Extraction Kit (QIAGEN) according tothe manufacturer's instructions. hparp2-Sal/Mlu was subcloned into thepTrcHis2™-Topo™ vector (Invitrogen) according to the manufacturer'sinstructions. The resultant plasmid, pTrcHis2-hparp2-Sal/Mlu, wasdigested with SalI and AvaII, and an hparp2 fragment (designatedhparp2-Sal/Ava) was isolated using gel electrophoresis and a QIAquick®Gel Extraction Kit (QIAGEN) and saved for further subcloning describedbelow.

[0292] A central region of hparp2 was isolated from hparp2 cDNA bydigesting with AvaII and SacI. The fragment (designated hparp2-Ava/Sac)was isolated using gel electrophoresis and a QIAquick Gel Extraction Kit(QIAGEN).

[0293] pFB-PARP2B-Sal/Sac, hparp2-Sal/Ava, and hparp2-Ava/Sac wereligated to produce pFB-hparp/Strep-tag. The Strep-tag was removed frompFB-hparp/Strep-tag by digestion with KpnI, yielding a plasmiddesignated pFB-hparp-Kpn. The KpnI digestion also removed the last 14amino acids of hPARP2. To replace this missing region, a fragment wasPCR amplified using a primer (5-P2-Kpn; SEQ ID NO:50) corresponding tothe sense strand of hparp2 polynucleotide sequence (nt 1757-1776 of SEQID NO:1) and a primer (3-P2-Kpn; SEQ ID NO:51) corresponding to theantisense strand of hparp2 polynucleotide sequence (nt 1799-1814 of SEQID NO:1). (SEQ ID NO:50) 5-P2-Kpn CCAGGTCCGTATGCGGTACC (SEQ ID NO:51)3-P2-Kpn GCCACGATGGGTACCGCGGCCGCTCACCACAGCTGAAGG

[0294] The PCR reaction contained 100 ng hparp2 cDNA, 0.5 μM eachprimer, 0.25 mM dNTPs, 1×PCR buffer, and 2.5 U of PfuTurbo® polymerasemix (Stratagene). The reactions were performed in a GeneAmp® PCR System9700 machine (PE Applied Biosystems) with the following steps: 1) 1cycle at 94° C. for 1 min; 2) 25 cycles of 94° C. for 30 sec, 55° C. for30 sec, and 72° C. for 30 sec; and 3) 1 cycle at 72° C. for 7 min. ThePCR fragment was digested with KpnI, isolated using gel electrophoresisand a QIAquick® Gel Extraction Kit (QIAGEN), and ligated withpFB-hparp-Kpn to produce pFB-hparp2.

[0295] pFB-hparp2 was sequenced with primers designed to anneal to thevector sequence (SEQ ID NOs:30 and 31) and primers designed to anneal tothe cDNA sequence (SEQ ID NOs:11, 12, 42, and 52-57).cDNA primers for pFB-hparp2 (SEQ ID NO:11) 3-P2-SEQ2CTTCCATGAGAGCTCGTCCATGCTGGCC (SEQ ID NO:12) 5-SEQ-2GGCCAGCATGGACGAGCTCTCATGGAAG (SEQ ID NO:42) AA#5 GTATTCTTTAGGCGAGAGGC(SEQ ID NO:52) AA#1 GGTGACGAAGTGGGCAGAAC (SEQ ID NO:53) AA#2TTCTGCCCACTTCGTCACCC (SEQ ID NO:54) AA#4 CGCAAGGCACAATGTAGGTT (SEQ IDNO:55) AA#6 GCCTCTCGCCTAAAGAATAC (SEQ ID NO:56) H2 AAGCAATCCTTCGGCCTTAG(SEQ ID NO:57) H6 AGTTCTGCCCACTTCGTCAC

[0296] The nucleotide sequence of pFB-hparp2 is set out in SEQ ID NO:58and the corresponding amino acid sequence of FB-hPARP2 is set out in SEQID NO:59. FB-hPARP2 contains a His tag leader region at aa 1-36. Aminoacids 37-619 of FB-hPARP2 represent full-length hPARP2.

[0297] Production of Recombinant Viral Stocks and Protein Purification

[0298] PARP1A/PARP2B and FB-hPARP2 recombinant viral stocks wereseparately produced using the FastBac system (Gibco BRL) according tothe manufacturer's suggested protocol and protein expression was carriedout as follows. Sf9 cells were grown at 27° C. in CCM3 medium (Hyclone,Logan, Utah) containing 50 U/mL penicillin and 50 μg/mL streptomycinsulfate (Gibco BRL). Exponentially growing cells were infected at amultiplicity of infection of approximately 0.5 virus per cell andincubated for 48 hr. Cells were collected by centrifugation at 1000×gfor 15 min, and the pellets were frozen and stored at −80° C. until use.

[0299] For protein purification, reagents were obtained from Sigma (St.Louis, Mo.), unless otherwise indicated. Cells were lysed in Lysisbuffer (25 mM Tris-HCl, pH 9.0, 50 mM glucose, 10 mM EDTA, 1 mM2-mercaptoethanol, 1 mM PMSF, 100 μM antipain, and 2 μg/mL aprotinin) bysonication. Igepal CA-630 (final concentration of 0.2%), Tween®-20(final concentration of 0.2%), and NaCl (final concentration of 0.5 M)were added to the Lysis buffer and the samples were agitated for 30 minat 4° C. The supernatants were collected after centrifugation at20,000×g for 20 min at 4° C. at which time they were treated with 1mg/mL protamine sulfate and allowed to stir for 1 hr at 4° C. Thesupernatants were collected after centrifugation at 4,000×g for 20 minat 4° C. at which time the protein was precipitated with 70% ammoniumsulfate for 1 hr at 4° C. Protein pellets were collected bycentrifugation at 20,000×g for 15 min at 4° C. and resuspended inRe-suspension buffer (100 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 10%glycerol, 1 mM PMSF, and 12 mM 2-mercaptoethanol).

[0300] Proteins were first purified via the His tag using TALON™Superflow Metal Affinity Resin (Clontech) and eluted with 200 mMimidazole (Clontech) according to the manufacturer's instructions. Theprotein elutions were next purified using a 3-aminobenzamide Affi-Gel®matrix (Bio-Rad Laboratories) prepared as described elsewhere [D'Amourset al., Anal Biochem 249:106-8 (1997)]. Proteins were eluted with 10 mM3-methoxybenzamide in Elution buffer (50 mM Tris-HCl, pH 7.5, 0.3 MNaCl, 10 mM 2-mercaptoethanol, 1 mM PMSF, 100 μM antipain, and 2 μg/mLaprotinin). The proteins were dialyzed 4× in 1 L Dialysis buffer (50 mMTris-HCl, pH 8.0, 1 mM dithiothreitol, 4 mM MgCl₂, 10 mM EDTA, 1 mMPMSF, and 2 μg/mL aprotinin). Glycerol was added to a finalconcentration of 10% and the proteins were stored at −80° C.

[0301] Poly(ADP-Ribose) Polymerase Activity

[0302] For poly(ADP-ribose) polymerase activity assays, reagents wereobtained from Sigma, unless otherwise indicated. PARP1A/PARP2B (250 ng)or FB-hPARP2 (25 ng) protein was incubated for 10 min at roomtemperature in assay buffer (total volume of 20 μL) containing 100 mMTris, pH 8.0, 1 mM MgCl₂, 10% glycerol, 1.5 mM dithiothreitol(Boehringer Mannheim/Roche Molecular Biochemicals, Indianapolis, Ind.),2.5 μM unlabeled NAD⁺, 16.7 μg/mL E. coli Strain B DNA, and 0.33 μCigamma-[³²P]-NAD⁺ (NEN, Boston, Mass.). Reactions were stopped by boilingin SDS running buffer and separated by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE). Autoradiography was used to visualizelabeled protein. Addition of poly(ADP-ribose) polymers to proteinsubstrate results in an increase in molecular weight of the protein, andsubsequently causes the protein to run higher on SDS-PAGE. Also, thelevel of poly(ADP-ribose) polymers added to the protein substrate canvary with each single protein molecule, resulting in labeled proteinwith different molecular weights and visualized on the autoradiographyfilm as a ladder or smear [for example, see Smith et al. (1998), supra].Both PARP1A/PARP2B and FB-hPARP2 possessed intrinsic poly(ADP-ribose)polymerase activity as shown by their ability produce poly(ADP-ribose)polymers. The PARP1A/PARP2B poly(ADP-ribose) polymerase reactionproduced a ladder of labeled protein approximately from 174 kDa to 200kDa. The FB-hPARP2 poly(ADP-ribose) polymerase reaction produced aladder of labeled protein approximately from 136 kDa to 250 kDa.

EXAMPLE 4

[0303] Preparation of Antibodies Immunoreactive with hPARP2 Polypeptides

[0304] The present invention provides for antibodies with specificityfor hPARP2 polypeptides. Antibodies to hPARP2 may be produced by anymethod known in the art, typically including, for example, theimmunization of laboratory animals with preparations of purified nativehPARP2, purified recombinant hPARP2, purified recombinant peptidefragments of hPARP2, or synthetic peptides derived from the hPARP2predicted amino acid sequence. In order to maximize the probability ofobtaining antibodies with appropriate specificity for hPARP2, regions ofthe polypeptide may be selected for use as an immunogen based upondifferences in those regions between hPARP1 and hPARP2. For example,amino acid residues 1-86 are substantially different between hPARP1 andhPARP2, and this region can be expressed as a truncated polypeptide inan appropriate expression system for use as an immunogen or to testpolyclonal or monoclonal antibody preparations. Similar approaches canbe applied to other regions of the hPARP2 polypeptide. Likewise,synthetic peptides can be made corresponding to selected regions ofhPARP2, and such peptides can be used to generate specific polyclonal ormonoclonal antibodies by methods known in the art [see, e.g., Harlow etal., supra].

[0305] Two regions in the carboxyl region of hPARP2 (designated hPARP2U1 and hPARP2 U2) were chosen as immunogens in antibody development.hparp2 U1 was amplified by PCR using a primer (5-PARP2 U1; SEQ ID NO:60)corresponding to the sense strand of hparp2 polynucleotide sequence (nt1074-1093 of SEQ ID NO:1) and a primer (PARP2 L2; SEQ ID NO:16)corresponding to the antisense strand of hparp2 polynucleotide sequence(nt 1790-1811 of SEQ ID NO:1). hparp2 U2 was amplified by PCR using aprimer (5-PARP2 U2; SEQ ID NO:61) corresponding to the sense strand ofhparp2 polynucleotide sequence (nt 1125-1145 of SEQ ID NO:1) and thesame antisense primer (PARP2 L2; SEQ ID NO:16) used for hparp2 U1.5-PARP2 U1 GGAGACATTGAAATTGCTAT (SEQ ID NO:60) 5-PARP2 U2GAACACCCATTGGACCAACAC (SEQ ID NO:61)

[0306] The PCR reaction contained 100 ng hparp2 cDNA, 0.5 μM eachprimer, 0.25 mM dNTPs, 1×PCR buffer, and 2.5 U of PfuTurbo® polymerasemix (Stratagene). The reactions were performed in a GeneAmp® PCR System9700 machine (PE Applied Biosystems) with the following steps: 1) 1cycle at 94° C. for 1 min; 2) 25 cycles of 94° C. for 30 sec, 55° C. for2 min, and 72° C. for 2 min; and 3) 1 cycle at 72° C. for 7 min. The PCRfragments were subcloned into the pTrcHis2™-Topo™ vector (Invitrogen)according to the manufacturer's instructions. hparp2 U1 and hparp2 U2were sequenced with primers designed to anneal to the vector (SEQ IDNOs:62 and 63). pTrcHis Forward GAGGTATATATTAATGTATCG (SEQ ID NO:62)pTrcHis Reverse GATTTAATCTGTATCAGG (SEQ ID NO:63)

[0307] The polynucleotide sequence of hparp2 U1 is set out in SEQ IDNO:64 and the amino acid sequence (hPARP2 U1) is set out in SEQ IDNO:65. hPARP2 U1 includes aa 338-583 of hPARP2 (SEQ ID NO:2). Thepolynucleotide sequence of hparp2 U2 is set out in SEQ ID NO:66 and theamino acid sequence (hPARP2 U2) is set out in SEQ ID NO:67. hPARP2 U2includes aa 355-583 of hPARP2 (SEQ ID NO:2).

[0308] hPARP2 U1 and hPARP2 U2 poly-His fusion proteins were expressedseparately in E. coli and were induced with 1 mM IPTG at 37° C. hPARP2U1 and hPARP2 U2 proteins were isolated from inclusion bodies usingB-PER™ Bacterial Protein Extraction Reagent (Pierce, Rockford, Ill.)according to the manufacturer's instructions.

[0309] Each of five 6 to 12 week old Balb/c mice were pre-bled on day 0and injected with 30 μg per mouse of a mixture of hPARP2 U1 and hPARP2U2 in Freund's complete adjuvant. Subsequent boosts were made on day 21and 63 in Freund's incomplete adjuvant. Mice were test bled on day 73and the bleeds were screened by ELISA. using standard methods, on platescoated with PARP1A/PARP2B (described above). Specific antibody wasdetected using goat anti-mouse IgG(fc) horseradish peroxidase conjugate.

[0310] ELISA reactive mouse sera are tested in Western analysis usingstandard methods. The spleens of mice reactive to FB-hPARP2 protein inthe Western analysis are removed and fused to NS-1 cells by standardmethods [Harlow and Lane, Antibodies, a Laboratory Manual, Cold SpringHarbor Laboratory (1988)] to produce monoclonal antibodies.

[0311] All publications and patent documents cited in this specificationare incorporated herein by reference for all that they disclose.

[0312] While the present invention has been described with specificreference to certain preferred embodiments for purposes of clarity andunderstanding, it will be apparent to the skilled artisan that furtherchanges and modifications may be practiced within the scope of theinvention as it is defined in the claims set forth below. Accordingly,no limitations should be placed on the invention other than thosespecifically recited in the claims.

1 68 1 1814 DNA Homo sapiens CDS (63)..(1811) 1 gcctagtgac actgggcccgcgattccttg gagcgggttg atgacgtcag cgttcgaatt 60 cc atg gcg gcg cgg cggcga cgg agc acc ggc ggc ggc agg gcg aga 107 Met Ala Ala Arg Arg Arg ArgSer Thr Gly Gly Gly Arg Ala Arg 1 5 10 15 gca tta aat gaa agc aaa agagtt aat aat ggc aac acg gct cca gaa 155 Ala Leu Asn Glu Ser Lys Arg ValAsn Asn Gly Asn Thr Ala Pro Glu 20 25 30 gac tct tcc cct gcc aag aaa actcgt aga tgc cag aga cag gag tcg 203 Asp Ser Ser Pro Ala Lys Lys Thr ArgArg Cys Gln Arg Gln Glu Ser 35 40 45 aaa aag atg cct gtg gct gga gga aaagct aat aag gac agg aca gaa 251 Lys Lys Met Pro Val Ala Gly Gly Lys AlaAsn Lys Asp Arg Thr Glu 50 55 60 gac aag caa gat ggt atg cca gga agg tcatgg gcc agc aaa agg gtc 299 Asp Lys Gln Asp Gly Met Pro Gly Arg Ser TrpAla Ser Lys Arg Val 65 70 75 tct gaa tct gtg aag gcc ttg ctg tta aag ggcaaa gct cct gtg gac 347 Ser Glu Ser Val Lys Ala Leu Leu Leu Lys Gly LysAla Pro Val Asp 80 85 90 95 cca gag tgt aca gcc aag gtg ggg aag gct catgtg tat tgt gaa gga 395 Pro Glu Cys Thr Ala Lys Val Gly Lys Ala His ValTyr Cys Glu Gly 100 105 110 aat gat gtc tat gat gtc atg cta aat cag accaat ctc cag ttc aac 443 Asn Asp Val Tyr Asp Val Met Leu Asn Gln Thr AsnLeu Gln Phe Asn 115 120 125 aac aac aag tac tat ctg att cag cta tta gaagat gat gcc cag agg 491 Asn Asn Lys Tyr Tyr Leu Ile Gln Leu Leu Glu AspAsp Ala Gln Arg 130 135 140 aac ttc agt gtt tgg atg aga tgg ggc cga gttggg aaa atg gga cag 539 Asn Phe Ser Val Trp Met Arg Trp Gly Arg Val GlyLys Met Gly Gln 145 150 155 cac agc ctg gtg gct tgt tca ggc aat ctc aacaag gcc aag gaa atc 587 His Ser Leu Val Ala Cys Ser Gly Asn Leu Asn LysAla Lys Glu Ile 160 165 170 175 ttt cag aag aaa ttc ctt gac aaa acg aaaaac aat tgg gaa gat cga 635 Phe Gln Lys Lys Phe Leu Asp Lys Thr Lys AsnAsn Trp Glu Asp Arg 180 185 190 gaa aag ttt gag aag gtg cct gga aaa tatgat atg cta cag atg gac 683 Glu Lys Phe Glu Lys Val Pro Gly Lys Tyr AspMet Leu Gln Met Asp 195 200 205 tat gcc acc aat act cag gat gaa gag gaaaca aag aaa gag gaa tct 731 Tyr Ala Thr Asn Thr Gln Asp Glu Glu Glu ThrLys Lys Glu Glu Ser 210 215 220 ctt aaa tct ccc ttg aag cca gag tca cagcta gat ctt cgg gta cag 779 Leu Lys Ser Pro Leu Lys Pro Glu Ser Gln LeuAsp Leu Arg Val Gln 225 230 235 gag tta ata aag ttg atc tgt aat gtt caggcc atg gaa gaa atg atg 827 Glu Leu Ile Lys Leu Ile Cys Asn Val Gln AlaMet Glu Glu Met Met 240 245 250 255 atg gaa atg aag tat aat acc aag aaagcc cca ctt ggg aag ctg aca 875 Met Glu Met Lys Tyr Asn Thr Lys Lys AlaPro Leu Gly Lys Leu Thr 260 265 270 gtg gca caa atc aag gca ggt tac cagtct ctt aag aag att gag gat 923 Val Ala Gln Ile Lys Ala Gly Tyr Gln SerLeu Lys Lys Ile Glu Asp 275 280 285 tgt att cgg gct ggc cag cat gga cgagct ctc atg gaa gca tgc aat 971 Cys Ile Arg Ala Gly Gln His Gly Arg AlaLeu Met Glu Ala Cys Asn 290 295 300 gaa ttc tac acc agg att ccg cat gacttt gga ctc cgt act cct cca 1019 Glu Phe Tyr Thr Arg Ile Pro His Asp PheGly Leu Arg Thr Pro Pro 305 310 315 cta atc cgg aca cag aag gaa ctg tcagaa aaa ata caa tta cta gag 1067 Leu Ile Arg Thr Gln Lys Glu Leu Ser GluLys Ile Gln Leu Leu Glu 320 325 330 335 gct ttg gga gac att gaa att gctatt aag ctg gtg aaa aca gag cta 1115 Ala Leu Gly Asp Ile Glu Ile Ala IleLys Leu Val Lys Thr Glu Leu 340 345 350 caa agc cca gaa cac cca ttg gaccaa cac tat aga aac cta cat tgt 1163 Gln Ser Pro Glu His Pro Leu Asp GlnHis Tyr Arg Asn Leu His Cys 355 360 365 gcc ttg cgc ccc ctt gac cat gaaagt tac gag ttc aaa gtg att tcc 1211 Ala Leu Arg Pro Leu Asp His Glu SerTyr Glu Phe Lys Val Ile Ser 370 375 380 cag tac cta caa tct acc cat gctccc aca cac agc gac tat acc atg 1259 Gln Tyr Leu Gln Ser Thr His Ala ProThr His Ser Asp Tyr Thr Met 385 390 395 acc ttg ctg gat ttg ttt gaa gtggag aag gat ggt gag aaa gaa gcc 1307 Thr Leu Leu Asp Leu Phe Glu Val GluLys Asp Gly Glu Lys Glu Ala 400 405 410 415 ttc aga gag gac ctt cat aacagg atg ctt cta tgg cat ggt tcc agg 1355 Phe Arg Glu Asp Leu His Asn ArgMet Leu Leu Trp His Gly Ser Arg 420 425 430 atg agt aac tgg gtg gga atcttg agc cat ggg ctt cga att gcc cca 1403 Met Ser Asn Trp Val Gly Ile LeuSer His Gly Leu Arg Ile Ala Pro 435 440 445 cct gaa gct ccc atc aca ggttac atg ttt ggg aaa gga atc tac ttt 1451 Pro Glu Ala Pro Ile Thr Gly TyrMet Phe Gly Lys Gly Ile Tyr Phe 450 455 460 gct gac atg tct tcc aag agtgcc aat tac tgc ttt gcc tct cgc cta 1499 Ala Asp Met Ser Ser Lys Ser AlaAsn Tyr Cys Phe Ala Ser Arg Leu 465 470 475 aag aat aca gga ctg ctg ctctta tca gag gta gct cta ggt cag tgt 1547 Lys Asn Thr Gly Leu Leu Leu LeuSer Glu Val Ala Leu Gly Gln Cys 480 485 490 495 aat gaa cta cta gag gccaat cct aag gcc gaa gga ttg ctt caa ggt 1595 Asn Glu Leu Leu Glu Ala AsnPro Lys Ala Glu Gly Leu Leu Gln Gly 500 505 510 aaa cat agc acc aag gggctg ggc aag atg gct ccc agt tct gcc cac 1643 Lys His Ser Thr Lys Gly LeuGly Lys Met Ala Pro Ser Ser Ala His 515 520 525 ttc gtc acc ctg aat gggagt aca gtg cca tta gga cca gca agt gac 1691 Phe Val Thr Leu Asn Gly SerThr Val Pro Leu Gly Pro Ala Ser Asp 530 535 540 aca gga att ctg aat ccagat ggt tat acc ctc aac tac aat gaa tat 1739 Thr Gly Ile Leu Asn Pro AspGly Tyr Thr Leu Asn Tyr Asn Glu Tyr 545 550 555 att gta tat aac ccc aaccag gtc cgt atg cgg tac ctt tta aag gtt 1787 Ile Val Tyr Asn Pro Asn GlnVal Arg Met Arg Tyr Leu Leu Lys Val 560 565 570 575 cag ttt aat ttc cttcag ctg tgg tga 1814 Gln Phe Asn Phe Leu Gln Leu Trp 580 2 583 PRT Homosapiens 2 Met Ala Ala Arg Arg Arg Arg Ser Thr Gly Gly Gly Arg Ala ArgAla 1 5 10 15 Leu Asn Glu Ser Lys Arg Val Asn Asn Gly Asn Thr Ala ProGlu Asp 20 25 30 Ser Ser Pro Ala Lys Lys Thr Arg Arg Cys Gln Arg Gln GluSer Lys 35 40 45 Lys Met Pro Val Ala Gly Gly Lys Ala Asn Lys Asp Arg ThrGlu Asp 50 55 60 Lys Gln Asp Gly Met Pro Gly Arg Ser Trp Ala Ser Lys ArgVal Ser 65 70 75 80 Glu Ser Val Lys Ala Leu Leu Leu Lys Gly Lys Ala ProVal Asp Pro 85 90 95 Glu Cys Thr Ala Lys Val Gly Lys Ala His Val Tyr CysGlu Gly Asn 100 105 110 Asp Val Tyr Asp Val Met Leu Asn Gln Thr Asn LeuGln Phe Asn Asn 115 120 125 Asn Lys Tyr Tyr Leu Ile Gln Leu Leu Glu AspAsp Ala Gln Arg Asn 130 135 140 Phe Ser Val Trp Met Arg Trp Gly Arg ValGly Lys Met Gly Gln His 145 150 155 160 Ser Leu Val Ala Cys Ser Gly AsnLeu Asn Lys Ala Lys Glu Ile Phe 165 170 175 Gln Lys Lys Phe Leu Asp LysThr Lys Asn Asn Trp Glu Asp Arg Glu 180 185 190 Lys Phe Glu Lys Val ProGly Lys Tyr Asp Met Leu Gln Met Asp Tyr 195 200 205 Ala Thr Asn Thr GlnAsp Glu Glu Glu Thr Lys Lys Glu Glu Ser Leu 210 215 220 Lys Ser Pro LeuLys Pro Glu Ser Gln Leu Asp Leu Arg Val Gln Glu 225 230 235 240 Leu IleLys Leu Ile Cys Asn Val Gln Ala Met Glu Glu Met Met Met 245 250 255 GluMet Lys Tyr Asn Thr Lys Lys Ala Pro Leu Gly Lys Leu Thr Val 260 265 270Ala Gln Ile Lys Ala Gly Tyr Gln Ser Leu Lys Lys Ile Glu Asp Cys 275 280285 Ile Arg Ala Gly Gln His Gly Arg Ala Leu Met Glu Ala Cys Asn Glu 290295 300 Phe Tyr Thr Arg Ile Pro His Asp Phe Gly Leu Arg Thr Pro Pro Leu305 310 315 320 Ile Arg Thr Gln Lys Glu Leu Ser Glu Lys Ile Gln Leu LeuGlu Ala 325 330 335 Leu Gly Asp Ile Glu Ile Ala Ile Lys Leu Val Lys ThrGlu Leu Gln 340 345 350 Ser Pro Glu His Pro Leu Asp Gln His Tyr Arg AsnLeu His Cys Ala 355 360 365 Leu Arg Pro Leu Asp His Glu Ser Tyr Glu PheLys Val Ile Ser Gln 370 375 380 Tyr Leu Gln Ser Thr His Ala Pro Thr HisSer Asp Tyr Thr Met Thr 385 390 395 400 Leu Leu Asp Leu Phe Glu Val GluLys Asp Gly Glu Lys Glu Ala Phe 405 410 415 Arg Glu Asp Leu His Asn ArgMet Leu Leu Trp His Gly Ser Arg Met 420 425 430 Ser Asn Trp Val Gly IleLeu Ser His Gly Leu Arg Ile Ala Pro Pro 435 440 445 Glu Ala Pro Ile ThrGly Tyr Met Phe Gly Lys Gly Ile Tyr Phe Ala 450 455 460 Asp Met Ser SerLys Ser Ala Asn Tyr Cys Phe Ala Ser Arg Leu Lys 465 470 475 480 Asn ThrGly Leu Leu Leu Leu Ser Glu Val Ala Leu Gly Gln Cys Asn 485 490 495 GluLeu Leu Glu Ala Asn Pro Lys Ala Glu Gly Leu Leu Gln Gly Lys 500 505 510His Ser Thr Lys Gly Leu Gly Lys Met Ala Pro Ser Ser Ala His Phe 515 520525 Val Thr Leu Asn Gly Ser Thr Val Pro Leu Gly Pro Ala Ser Asp Thr 530535 540 Gly Ile Leu Asn Pro Asp Gly Tyr Thr Leu Asn Tyr Asn Glu Tyr Ile545 550 555 560 Val Tyr Asn Pro Asn Gln Val Arg Met Arg Tyr Leu Leu LysVal Gln 565 570 575 Phe Asn Phe Leu Gln Leu Trp 580 3 472 DNA Homosapiens 3 tttttttttt ttttttttag acctgtacag tttttattac ataaaatatcacaaaattca 60 caagtacaac actgcttatt ttcttgcttg aagatcagat ctctggtttatttaagatca 120 acattcacca cagctgaagg aaattaaact gaacctttaa aaggtaccgcatacggacct 180 ggttggggtt atatacaata tattcattgt agttgagggt ataaccatctggattcagaa 240 ttcctgtgtc acttgctggt cctaatggca ctgtactccc attcctgccaaatggaaaaa 300 aagtgtgtca acatcagtct ctggttcaga agctgcaata gagaacgtagtcttatctgg 360 ccaaaaggag tcttctagtc ctcctggttc tgagtactta cagggtgacgaagtgggcag 420 aactgggagc catcttgccc agccccttgg ggctatgttt accttgaagc aa472 4 476 DNA Homo sapiens misc_feature (4) n = A or T or G or C 4aganctgtac agtttttatt acataaaata tcacaaaatt cacaagtaca cactgcttat 60tttcttgctt gaagatcaga tctctggttt atttaatatc aacattcacc acagctgaag 120gaaattaaac tgaaccttta aaaggtaccg catacggacc tgggttgggg ttatatacaa 180tatattcatt gtagttgagg gtataacatc tgggattcag aattcctgtg tcacttgctg 240ggncctaatg ggcactgtac tcccattcag gggtgacgag tgggggcagg aactggggag 300gccatcttgc ccaggcccct tgggngctat ggtttacctt gaaggcaatc cttcgggcct 360tagggattgg gcctctagta gttcattaca ctggacctag gggctacctc tggtaggggc 420agcagtcccg tattttttag ggcnagaggg naaagcagtt attngggcan ttttgg 476 5 416DNA Homo sapiens misc_feature (334) n = A or T or G or C 5 gctttgggagacattgaaat tgctattaag ctggtgaaaa cagagctaca aagcccagaa 60 cacccattggaccaacacta tagaaaccta cattgtgcct tgcgccccct tgaccatgaa 120 agttatgagttcaaagtgat ttcccagtac ctacaatcta cccatgctcc cacacacagc 180 gactattaccatggaccttg ctgggatttg tttgaagtgg gaggaaggga tgggtgagga 240 aaggaaggcctttcaggagg agggaccttt cattaacagg gatgctttct atggggcatg 300 ggttccaggggttgaggtaa ctggggttgg ggantctttg aggccntggg gttttcggan 360 tttgcccccaccttggaagg ntccccntca cagggtttac atgtttttgg gggaaa 416 6 38 DNAArtificial Sequence Description of Artificial Sequence Primer 6gtgttggtcc aatgggtgtt ctgggctttg tagctctg 38 7 27 DNA ArtificialSequence Description of Artificial Sequence Primer 7 ccatcctaatacgactcact atagggc 27 8 18 DNA Artificial Sequence Description ofArtificial Sequence Primer 8 tgtaaaacga cggccagt 18 9 19 DNA ArtificialSequence Description of Artificial Sequence Primer 9 ggaaacagctatgaccatg 19 10 27 DNA Artificial Sequence Description of ArtificialSequencePrimer 10 ggctgtacac tctgggtcca caggagc 27 11 28 DNA ArtificialSequence Description of Artificial SequencePrimer 11 cttccatgagagctcgtcca tgctggcc 28 12 28 DNA Artificial Sequence Description ofArtificial SequencePrimer 12 ggccagcatg gacgagctct catggaag 28 13 1143DNA Homo sapiens 13 gcctagtgac actgggcccg cgattccttg gagcgggttgatgacgtcag cgttcgaatt 60 ccatggcggc gcggcggcga cggagcaccg gcggcggcagggcgagagca ttaaatgaaa 120 gcaaaagagt taataatggc aacacggctc cagaagactcttcccctgcc aagaaaactc 180 gtagatgcca gagacaggag tcgaaaaaga tgcctgtggctggaggaaaa gctaataagg 240 acaggacaga agacaagcaa gatggtatgc caggaaggtcatgggccagc aaaagggtct 300 ctgaatctgt gaaggccttg ctgttaaagg gcaaagctcctgtggaccca gagtgtacag 360 ccaaggtggg gaaggctcat gtgtattgtg aaggaaatgatgtctatgat gtcatgctaa 420 atcagaccaa tctccagttc aacaacaaca agtactatctgattcagcta ttagaagatg 480 atgcccagag gaacttcagt gtttggatga gatggggccgagttgggaaa atgggacagc 540 acagcctggt ggcttgttca ggcaatctca acaaggccaaggaaatcttt cagaagaaat 600 tccttgacaa aacgaaaaac aattgggaag atcgagaaaagtttgagaag gtgcctggaa 660 aatatgatat gctacagatg gactatgcca ccaatactcaggatgaagag gaaacaaaga 720 aagaggaatc tcttaaatct cccttgaagc cagagtcacagctagatctt cgggtacagg 780 agttaataaa gttgatctgt aatgttcagg ccatggaagaaatgatgatg gaaatgaagt 840 ataataccaa gaaagcccca cttgggaagc tgacagtggcacaaatcaag gcaggttacc 900 agtctcttaa gaagattgag gattgtattc gggctggccagcatggacga gctctcatgg 960 aagcatgcaa tgaattctac accaggattc cgcatgactttggactccgt actcctccac 1020 taatccggac acagaaggaa ctgtcagaaa aaatacaattactagaggct ttgggagaca 1080 ttgaaattgc tattaagctg gtgaaaacag agctacaaagcccagaacac ccattggacc 1140 aac 1143 14 27 DNA Artificial SequenceDescription of Artificial Sequence Primer 14 gctcctgtgg acccagagtgtacagcc 27 15 20 DNA Artificial Sequence Description of ArtificialSequence Primer 15 acattcacca cagctgaagg 20 16 22 DNA ArtificialSequence Description of Artificial Sequence Primer 16 ccacagctgaaggaaattaa ac 22 17 550 DNA Artificial Sequence Description ofArtificial SequenceP2-1 (hPARP2 Fragment) 17 agccaaggtg gggaaggctcatgtgtattg tgaaggaaat gatgtctatg atgtcatgct 60 aaatcagacc aatctccagttcaacaacaa caagtactat ctgattcagc tattagaaga 120 tgatgcccag aggaacttcagtgtttggat gagatggggc cgagttggga aaatgggaca 180 gcacagcctg gtggcttgttcaggcaatct caacaaggcc aaggaaatct ttcagaagaa 240 attccttgac aaaacgaaaaacaattggga agatcgagaa aagtttgaga aggtgcctgg 300 aaaatatgat atgctacagatggactatgc caccaatact caggatgaag aggaaacaaa 360 gaaagaggaa tctcttaaatctcccttgaa gccagagtca cagctagatc ttcgggtaca 420 ggagttaata aagttgatctgtaatgttca ggccatggaa gaaatgatga tggaaatgaa 480 gtataatacc aagaaagccccacttgggaa gctgacagtg gcacaaatca aggcaggtta 540 ccagtctctt 550 18 550DNA Artificial Sequence Description of Artificial SequenceP2-9 (hPARP2Fragment) 18 agccaaggtg gggaaggctc atgtgtattg tgaaggaaat gatgtctatgatgtcatgct 60 aaatcagacc aatctccagt tcaacaacaa caagtactat ctgattcagctattagaaga 120 tgatgcccag aggaacttca gtgtttggat gagatggggc cgagttgggaaaatgggaca 180 gcacagcctg gtggcttgtt caggcaatct caacaaggcc aaggaaatctttcagaagaa 240 attccttgac aaaacgaaaa acaattggga agatcgagaa aagtttgagaaggtgcctgg 300 aaaatatgat atgctacaga tggactatgc caccaatact caggatgaagaggaaacaaa 360 gaaagaggaa tctcttaaat ctcccttgaa gccagagtca cagctagatcttcgggtaca 420 ggagttaata aagttgatct gtaatgttca ggccatggaa gaaatgatgatggaaatgaa 480 gtataatacc aagaaagccc cacttgggaa gctgacagtg gcacaaatcaaggcaggtta 540 ccagtctctt 550 19 360 PRT Homo sapiens 19 Met Ala Ala ArgArg Arg Arg Ser Thr Gly Gly Gly Arg Ala Arg Ala 1 5 10 15 Leu Asn GluSer Lys Arg Val Asn Asn Gly Asn Thr Ala Pro Glu Asp 20 25 30 Ser Ser ProAla Lys Lys Thr Arg Arg Cys Gln Arg Gln Glu Ser Lys 35 40 45 Lys Met ProVal Ala Gly Gly Lys Ala Asn Lys Asp Arg Thr Glu Asp 50 55 60 Lys Gln AspGly Met Pro Gly Arg Ser Trp Ala Ser Lys Arg Val Ser 65 70 75 80 Glu SerVal Lys Ala Leu Leu Leu Lys Gly Lys Ala Pro Val Asp Pro 85 90 95 Glu CysThr Ala Lys Val Gly Lys Ala His Val Tyr Cys Glu Gly Asn 100 105 110 AspVal Tyr Asp Val Met Leu Asn Gln Thr Asn Leu Gln Phe Asn Asn 115 120 125Asn Lys Tyr Tyr Leu Ile Gln Leu Leu Glu Asp Asp Ala Gln Arg Asn 130 135140 Phe Ser Val Trp Met Arg Trp Gly Arg Val Gly Lys Met Gly Gln His 145150 155 160 Ser Leu Val Ala Cys Ser Gly Asn Leu Asn Lys Ala Lys Glu IlePhe 165 170 175 Gln Lys Lys Phe Leu Asp Lys Thr Lys Asn Asn Trp Glu AspArg Glu 180 185 190 Lys Phe Glu Lys Val Pro Gly Lys Tyr Asp Met Leu GlnMet Asp Tyr 195 200 205 Ala Thr Asn Thr Gln Asp Glu Glu Glu Thr Lys LysGlu Glu Ser Leu 210 215 220 Lys Ser Pro Leu Lys Pro Glu Ser Gln Leu AspLeu Arg Val Gln Glu 225 230 235 240 Leu Ile Lys Leu Ile Cys Asn Val GlnAla Met Glu Glu Met Met Met 245 250 255 Glu Met Lys Tyr Asn Thr Lys LysAla Pro Leu Gly Lys Leu Thr Val 260 265 270 Ala Gln Ile Lys Ala Gly TyrGln Ser Leu Lys Lys Ile Glu Asp Cys 275 280 285 Ile Arg Ala Gly Gln HisGly Arg Ala Leu Met Glu Ala Cys Asn Glu 290 295 300 Phe Tyr Thr Arg IlePro His Asp Phe Gly Leu Arg Thr Pro Pro Leu 305 310 315 320 Ile Arg ThrGln Lys Glu Leu Ser Glu Lys Ile Gln Leu Leu Glu Ala 325 330 335 Leu GlyAsp Ile Glu Ile Ala Ile Lys Leu Val Lys Thr Glu Leu Gln 340 345 350 SerPro Glu His Pro Leu Asp Gln 355 360 20 864 DNA Homo sapiens 20gctctcatgg aagcatgcaa tgaattctac accaggattc cgcatgactt tggactccgt 60actcctccac taatccggac acagaaggaa ctgtcagaaa aaatacaatt actagaggct 120ttgggagaca ttgaaattgc tattaagctg gtgaaaacag agctacaaag cccagaacac 180ccattggacc aacactatag aaacctacat tgtgccttgc gcccccttga ccatgaaagt 240tacgagttca aagtgatttc ccagtaccta caatctaccc atgctcccac acacagcgac 300tataccatga ccttgctgga tttgtttgaa gtggagaagg atggtgagaa agaagccttc 360agagaggacc ttcataacag gatgcttcta tggcatggtt ccaggatgag taactgggtg 420ggaatcttga gccatgggct tcgaattgcc ccacctgaag ctcccatcac aggttacatg 480tttgggaaag gaatctactt tgctgacatg tcttccaaga gtgccaatta ctgctttgcc 540tctcgcctaa agaatacagg actgctgctc ttatcagagg tagctctagg tcagtgtaat 600gaactactag aggccaatcc taaggccgaa ggattgcttc aaggtaaaca tagcaccaag 660gggctgggca agatggctcc cagttctgcc cacttcgtca ccctgaatgg gagtacagtg 720ccattaggac cagcaagtga cacaggaatt ctgaatccag atggttatac cctcaactac 780aatgaatata ttgtatataa ccccaaccag gtccgtatgc ggtacctttt aaaggttcag 840tttaatttcc ttcagctgtg gtga 864 21 287 PRT Homo sapiens 21 Ala Leu MetGlu Ala Cys Asn Glu Phe Tyr Thr Arg Ile Pro His Asp 1 5 10 15 Phe GlyLeu Arg Thr Pro Pro Leu Ile Arg Thr Gln Lys Glu Leu Ser 20 25 30 Glu LysIle Gln Leu Leu Glu Ala Leu Gly Asp Ile Glu Ile Ala Ile 35 40 45 Lys LeuVal Lys Thr Glu Leu Gln Ser Pro Glu His Pro Leu Asp Gln 50 55 60 His TyrArg Asn Leu His Cys Ala Leu Arg Pro Leu Asp His Glu Ser 65 70 75 80 TyrGlu Phe Lys Val Ile Ser Gln Tyr Leu Gln Ser Thr His Ala Pro 85 90 95 ThrHis Ser Asp Tyr Thr Met Thr Leu Leu Asp Leu Phe Glu Val Glu 100 105 110Lys Asp Gly Glu Lys Glu Ala Phe Arg Glu Asp Leu His Asn Arg Met 115 120125 Leu Leu Trp His Gly Ser Arg Met Ser Asn Trp Val Gly Ile Leu Ser 130135 140 His Gly Leu Arg Ile Ala Pro Pro Glu Ala Pro Ile Thr Gly Tyr Met145 150 155 160 Phe Gly Lys Gly Ile Tyr Phe Ala Asp Met Ser Ser Lys SerAla Asn 165 170 175 Tyr Cys Phe Ala Ser Arg Leu Lys Asn Thr Gly Leu LeuLeu Leu Ser 180 185 190 Glu Val Ala Leu Gly Gln Cys Asn Glu Leu Leu GluAla Asn Pro Lys 195 200 205 Ala Glu Gly Leu Leu Gln Gly Lys His Ser ThrLys Gly Leu Gly Lys 210 215 220 Met Ala Pro Ser Ser Ala His Phe Val ThrLeu Asn Gly Ser Thr Val 225 230 235 240 Pro Leu Gly Pro Ala Ser Asp ThrGly Ile Leu Asn Pro Asp Gly Tyr 245 250 255 Thr Leu Asn Tyr Asn Glu TyrIle Val Tyr Asn Pro Asn Gln Val Arg 260 265 270 Met Arg Tyr Leu Leu LysVal Gln Phe Asn Phe Leu Gln Leu Trp 275 280 285 22 1707 DNA Mus musculusCDS (16)..(1695) 22 ctcgagtcaa gagcg atg gcg ccg cgg cgg cag aga tca ggctct gga agg 51 Met Ala Pro Arg Arg Gln Arg Ser Gly Ser Gly Arg 1 5 10cga gtg cta aat gaa gcc aag aaa gtt gat aat ggc aac aaa gca aca 99 ArgVal Leu Asn Glu Ala Lys Lys Val Asp Asn Gly Asn Lys Ala Thr 15 20 25 gaagac gac tct cct cct ggc aag aag atg cgc acg tgc cag aga aaa 147 Glu AspAsp Ser Pro Pro Gly Lys Lys Met Arg Thr Cys Gln Arg Lys 30 35 40 ggg cctatg gct gga ggg aag gac gca gac agg aca aaa gac aat cga 195 Gly Pro MetAla Gly Gly Lys Asp Ala Asp Arg Thr Lys Asp Asn Arg 45 50 55 60 gac tctgtg aag acc ttg ctg tta aag ggc aaa gcc cct gtg gac cca 243 Asp Ser ValLys Thr Leu Leu Leu Lys Gly Lys Ala Pro Val Asp Pro 65 70 75 gag tgt gcagcc aag ctg gga aag gct cat gtg tat tgt gaa gga gat 291 Glu Cys Ala AlaLys Leu Gly Lys Ala His Val Tyr Cys Glu Gly Asp 80 85 90 gat gtc tat gatgtc atg cta aat caa acc aat ctc cag ttc aac aac 339 Asp Val Tyr Asp ValMet Leu Asn Gln Thr Asn Leu Gln Phe Asn Asn 95 100 105 aac aag tac tacctt att cag ctg tta gaa gat gat gcc cag agg aac 387 Asn Lys Tyr Tyr LeuIle Gln Leu Leu Glu Asp Asp Ala Gln Arg Asn 110 115 120 ttc agt gtt tggatg agg tgg ggc cga gtt gga aag acg ggg cag cac 435 Phe Ser Val Trp MetArg Trp Gly Arg Val Gly Lys Thr Gly Gln His 125 130 135 140 agc ttg gtgact tgt tct ggt gac ctc aac aaa gca aaa gaa ata ttt 483 Ser Leu Val ThrCys Ser Gly Asp Leu Asn Lys Ala Lys Glu Ile Phe 145 150 155 cag aaa aaattc ctt gac aaa act aaa aac aat tgg gag gac cgt gag 531 Gln Lys Lys PheLeu Asp Lys Thr Lys Asn Asn Trp Glu Asp Arg Glu 160 165 170 aac ttt gaaaaa gta cct gga aaa tac gac atg tta cag atg gac tat 579 Asn Phe Glu LysVal Pro Gly Lys Tyr Asp Met Leu Gln Met Asp Tyr 175 180 185 gct gcc agcacg cag gat gaa agt aaa aca aaa gaa gag gaa act ttg 627 Ala Ala Ser ThrGln Asp Glu Ser Lys Thr Lys Glu Glu Glu Thr Leu 190 195 200 aag cct gagtct cag ctg gat ctt cga gtc cag gag ctg cta aag ttg 675 Lys Pro Glu SerGln Leu Asp Leu Arg Val Gln Glu Leu Leu Lys Leu 205 210 215 220 atc tgtaac gtg cag acc atg gaa gaa atg atg att gag atg aag tat 723 Ile Cys AsnVal Gln Thr Met Glu Glu Met Met Ile Glu Met Lys Tyr 225 230 235 gac accaag aga gcc ccg ctt gga aag ctg aca gtg gcg caa atc aag 771 Asp Thr LysArg Ala Pro Leu Gly Lys Leu Thr Val Ala Gln Ile Lys 240 245 250 gcc ggttac cag tct ctc aag aag att gag gac tgc atc cgc gct ggc 819 Ala Gly TyrGln Ser Leu Lys Lys Ile Glu Asp Cys Ile Arg Ala Gly 255 260 265 cag catggg cga gcg ctt gtt gaa gcg tgc aat gaa ttc tac acc agg 867 Gln His GlyArg Ala Leu Val Glu Ala Cys Asn Glu Phe Tyr Thr Arg 270 275 280 atc cctcat gac ttt gga ctc tcc atc cct cca gta atc cgg aca gag 915 Ile Pro HisAsp Phe Gly Leu Ser Ile Pro Pro Val Ile Arg Thr Glu 285 290 295 300 aaggaa ctg tca gac aaa gta aaa ctg cta gag gca ttg gga gac att 963 Lys GluLeu Ser Asp Lys Val Lys Leu Leu Glu Ala Leu Gly Asp Ile 305 310 315 gaaatt gcc ctt aaa ctg gtg aag tca gag cgc caa ggc cta gaa cac 1011 Glu IleAla Leu Lys Leu Val Lys Ser Glu Arg Gln Gly Leu Glu His 320 325 330 ccactg gac caa cac tat aga aac cta cac tgt gct ttg cgt cct ctg 1059 Pro LeuAsp Gln His Tyr Arg Asn Leu His Cys Ala Leu Arg Pro Leu 335 340 345 gaccat gaa agt aat gag ttt aag gtg att tct cag tac cta cag tct 1107 Asp HisGlu Ser Asn Glu Phe Lys Val Ile Ser Gln Tyr Leu Gln Ser 350 355 360 acgcat gct cct aca cac aag gac tat act atg acc ttg ctg gat gtt 1155 Thr HisAla Pro Thr His Lys Asp Tyr Thr Met Thr Leu Leu Asp Val 365 370 375 380ttc gaa gta gag aag gaa ggg gag aaa gag gcc ttc agg gag gac ctt 1203 PheGlu Val Glu Lys Glu Gly Glu Lys Glu Ala Phe Arg Glu Asp Leu 385 390 395cct aac agg atg ctg ctc tgg cat gga tcc agg ctg agt aac tgg gtg 1251 ProAsn Arg Met Leu Leu Trp His Gly Ser Arg Leu Ser Asn Trp Val 400 405 410ggg atc ctg agc cac ggg ctt aga gtt gcc cca cct gag gct ccc atc 1299 GlyIle Leu Ser His Gly Leu Arg Val Ala Pro Pro Glu Ala Pro Ile 415 420 425aca ggt tat atg ttt gga aaa gga atc tac ttt gct gac atg tcc tcc 1347 ThrGly Tyr Met Phe Gly Lys Gly Ile Tyr Phe Ala Asp Met Ser Ser 430 435 440aag agt gcc aat tac tgc ttt gcc tct cgc cta aag aat aca gga ttg 1395 LysSer Ala Asn Tyr Cys Phe Ala Ser Arg Leu Lys Asn Thr Gly Leu 445 450 455460 ctt ctt ctg tca gag gta gct cta ggt cag tgt aat gaa cta ctg gag 1443Leu Leu Leu Ser Glu Val Ala Leu Gly Gln Cys Asn Glu Leu Leu Glu 465 470475 gcc aat cct aaa gca caa gga ttg ctt cgg ggc aag cat agc acc aag 1491Ala Asn Pro Lys Ala Gln Gly Leu Leu Arg Gly Lys His Ser Thr Lys 480 485490 ggg atg gga aag atg gct ccc agc cct gcc cac ttc atc acc ctg aat 1539Gly Met Gly Lys Met Ala Pro Ser Pro Ala His Phe Ile Thr Leu Asn 495 500505 ggg agt aca gtg ccc tta gga cca gca agt gac aca gga att ctc aat 1587Gly Ser Thr Val Pro Leu Gly Pro Ala Ser Asp Thr Gly Ile Leu Asn 510 515520 cca gag ggg tac acc ctc aac tac aat gag ttt att gtt tat agc ccc 1635Pro Glu Gly Tyr Thr Leu Asn Tyr Asn Glu Phe Ile Val Tyr Ser Pro 525 530535 540 aac cag gtc cgt atg cga tac ctt cta aag att caa ttt aac ttc ctg1683 Asn Gln Val Arg Met Arg Tyr Leu Leu Lys Ile Gln Phe Asn Phe Leu 545550 555 cag cta tgg tga atgttgctcg ag 1707 Gln Leu Trp 23 559 PRT Musmusculus 23 Met Ala Pro Arg Arg Gln Arg Ser Gly Ser Gly Arg Arg Val LeuAsn 1 5 10 15 Glu Ala Lys Lys Val Asp Asn Gly Asn Lys Ala Thr Glu AspAsp Ser 20 25 30 Pro Pro Gly Lys Lys Met Arg Thr Cys Gln Arg Lys Gly ProMet Ala 35 40 45 Gly Gly Lys Asp Ala Asp Arg Thr Lys Asp Asn Arg Asp SerVal Lys 50 55 60 Thr Leu Leu Leu Lys Gly Lys Ala Pro Val Asp Pro Glu CysAla Ala 65 70 75 80 Lys Leu Gly Lys Ala His Val Tyr Cys Glu Gly Asp AspVal Tyr Asp 85 90 95 Val Met Leu Asn Gln Thr Asn Leu Gln Phe Asn Asn AsnLys Tyr Tyr 100 105 110 Leu Ile Gln Leu Leu Glu Asp Asp Ala Gln Arg AsnPhe Ser Val Trp 115 120 125 Met Arg Trp Gly Arg Val Gly Lys Thr Gly GlnHis Ser Leu Val Thr 130 135 140 Cys Ser Gly Asp Leu Asn Lys Ala Lys GluIle Phe Gln Lys Lys Phe 145 150 155 160 Leu Asp Lys Thr Lys Asn Asn TrpGlu Asp Arg Glu Asn Phe Glu Lys 165 170 175 Val Pro Gly Lys Tyr Asp MetLeu Gln Met Asp Tyr Ala Ala Ser Thr 180 185 190 Gln Asp Glu Ser Lys ThrLys Glu Glu Glu Thr Leu Lys Pro Glu Ser 195 200 205 Gln Leu Asp Leu ArgVal Gln Glu Leu Leu Lys Leu Ile Cys Asn Val 210 215 220 Gln Thr Met GluGlu Met Met Ile Glu Met Lys Tyr Asp Thr Lys Arg 225 230 235 240 Ala ProLeu Gly Lys Leu Thr Val Ala Gln Ile Lys Ala Gly Tyr Gln 245 250 255 SerLeu Lys Lys Ile Glu Asp Cys Ile Arg Ala Gly Gln His Gly Arg 260 265 270Ala Leu Val Glu Ala Cys Asn Glu Phe Tyr Thr Arg Ile Pro His Asp 275 280285 Phe Gly Leu Ser Ile Pro Pro Val Ile Arg Thr Glu Lys Glu Leu Ser 290295 300 Asp Lys Val Lys Leu Leu Glu Ala Leu Gly Asp Ile Glu Ile Ala Leu305 310 315 320 Lys Leu Val Lys Ser Glu Arg Gln Gly Leu Glu His Pro LeuAsp Gln 325 330 335 His Tyr Arg Asn Leu His Cys Ala Leu Arg Pro Leu AspHis Glu Ser 340 345 350 Asn Glu Phe Lys Val Ile Ser Gln Tyr Leu Gln SerThr His Ala Pro 355 360 365 Thr His Lys Asp Tyr Thr Met Thr Leu Leu AspVal Phe Glu Val Glu 370 375 380 Lys Glu Gly Glu Lys Glu Ala Phe Arg GluAsp Leu Pro Asn Arg Met 385 390 395 400 Leu Leu Trp His Gly Ser Arg LeuSer Asn Trp Val Gly Ile Leu Ser 405 410 415 His Gly Leu Arg Val Ala ProPro Glu Ala Pro Ile Thr Gly Tyr Met 420 425 430 Phe Gly Lys Gly Ile TyrPhe Ala Asp Met Ser Ser Lys Ser Ala Asn 435 440 445 Tyr Cys Phe Ala SerArg Leu Lys Asn Thr Gly Leu Leu Leu Leu Ser 450 455 460 Glu Val Ala LeuGly Gln Cys Asn Glu Leu Leu Glu Ala Asn Pro Lys 465 470 475 480 Ala GlnGly Leu Leu Arg Gly Lys His Ser Thr Lys Gly Met Gly Lys 485 490 495 MetAla Pro Ser Pro Ala His Phe Ile Thr Leu Asn Gly Ser Thr Val 500 505 510Pro Leu Gly Pro Ala Ser Asp Thr Gly Ile Leu Asn Pro Glu Gly Tyr 515 520525 Thr Leu Asn Tyr Asn Glu Phe Ile Val Tyr Ser Pro Asn Gln Val Arg 530535 540 Met Arg Tyr Leu Leu Lys Ile Gln Phe Asn Phe Leu Gln Leu Trp 545550 555 24 3045 DNA Homo sapiens CDS (1)..(3045) 24 atg gcg gag tct tcggat aag ctc tat cga gtc gag tac gcc aag agc 48 Met Ala Glu Ser Ser AspLys Leu Tyr Arg Val Glu Tyr Ala Lys Ser 1 5 10 15 ggg cgc gcc tct tgcaag aaa tgc agc gag agc atc ccc aag gac tcg 96 Gly Arg Ala Ser Cys LysLys Cys Ser Glu Ser Ile Pro Lys Asp Ser 20 25 30 ctc cgg atg gcc atc atggtg cag tcg ccc atg ttt gat gga aaa gtc 144 Leu Arg Met Ala Ile Met ValGln Ser Pro Met Phe Asp Gly Lys Val 35 40 45 cca cac tgg tac cac ttc tcctgc ttc tgg aag gtg ggc cac tcc atc 192 Pro His Trp Tyr His Phe Ser CysPhe Trp Lys Val Gly His Ser Ile 50 55 60 cgg cac cct gac gtt gag gtg gatggg ttc tct gag ctt cgg tgg gat 240 Arg His Pro Asp Val Glu Val Asp GlyPhe Ser Glu Leu Arg Trp Asp 65 70 75 80 gac cag cag aaa gtc aag aag acagcg gaa gct gga gga gtg aca ggc 288 Asp Gln Gln Lys Val Lys Lys Thr AlaGlu Ala Gly Gly Val Thr Gly 85 90 95 aaa ggc cag gat gga att ggt agc aaggca gag aag act ctg ggt gac 336 Lys Gly Gln Asp Gly Ile Gly Ser Lys AlaGlu Lys Thr Leu Gly Asp 100 105 110 ttt gca gca gag tat gcc aag tcc aacaga agt acg tgc aag ggg tgt 384 Phe Ala Ala Glu Tyr Ala Lys Ser Asn ArgSer Thr Cys Lys Gly Cys 115 120 125 atg gag aag ata gaa aag ggc cag gtgcgc ctg tcc aag aag atg gtg 432 Met Glu Lys Ile Glu Lys Gly Gln Val ArgLeu Ser Lys Lys Met Val 130 135 140 gac ccg gag aag cca cag cta ggc atgatt gac cgc tgg tac cat cca 480 Asp Pro Glu Lys Pro Gln Leu Gly Met IleAsp Arg Trp Tyr His Pro 145 150 155 160 ggc tgc ttt gtc aag aac agg gaggag ctg ggt ttc cgg ccc gag tac 528 Gly Cys Phe Val Lys Asn Arg Glu GluLeu Gly Phe Arg Pro Glu Tyr 165 170 175 agt gcg agt cag ctc aag ggc ttcagc ctc ctt gct aca gag gat aaa 576 Ser Ala Ser Gln Leu Lys Gly Phe SerLeu Leu Ala Thr Glu Asp Lys 180 185 190 gaa gcc ctg aag aag cag ctc ccagga gtc aag agt gaa gga aag aga 624 Glu Ala Leu Lys Lys Gln Leu Pro GlyVal Lys Ser Glu Gly Lys Arg 195 200 205 aaa ggc gat gag gtg gat gga gtggat gaa gtg gcg aag aag aaa tct 672 Lys Gly Asp Glu Val Asp Gly Val AspGlu Val Ala Lys Lys Lys Ser 210 215 220 aaa aaa gaa aaa gac aag gat agtaag ctt gaa aaa gcc cta aag gct 720 Lys Lys Glu Lys Asp Lys Asp Ser LysLeu Glu Lys Ala Leu Lys Ala 225 230 235 240 cag aac gac ctg atc tgg aacatc aag gac gag cta aag aaa gtg tgt 768 Gln Asn Asp Leu Ile Trp Asn IleLys Asp Glu Leu Lys Lys Val Cys 245 250 255 tca act aat gac ctg aag gagcta ctc atc ttc aac aag cag caa gtg 816 Ser Thr Asn Asp Leu Lys Glu LeuLeu Ile Phe Asn Lys Gln Gln Val 260 265 270 cct tct ggg gag tcg gcg atcttg gac cga gta gct gat ggc atg gtg 864 Pro Ser Gly Glu Ser Ala Ile LeuAsp Arg Val Ala Asp Gly Met Val 275 280 285 ttc ggt gcc ctc ctt ccc tgcgag gaa tgc tcg ggt cag ctg gtc ttc 912 Phe Gly Ala Leu Leu Pro Cys GluGlu Cys Ser Gly Gln Leu Val Phe 290 295 300 aag agc gat gcc tat tac tgcact ggg gac gtc act gcc tgg acc aag 960 Lys Ser Asp Ala Tyr Tyr Cys ThrGly Asp Val Thr Ala Trp Thr Lys 305 310 315 320 tgt atg gtc aag aca cagaca ccc aac cgg aag gag tgg gta acc cca 1008 Cys Met Val Lys Thr Gln ThrPro Asn Arg Lys Glu Trp Val Thr Pro 325 330 335 aag gaa ttc cga gaa atctct tac ctc aag aaa ttg aag gtt aaa aag 1056 Lys Glu Phe Arg Glu Ile SerTyr Leu Lys Lys Leu Lys Val Lys Lys 340 345 350 cag gac cgt ata ttc ccccca gaa acc agc gcc tcc gtg gcg gcc acg 1104 Gln Asp Arg Ile Phe Pro ProGlu Thr Ser Ala Ser Val Ala Ala Thr 355 360 365 cct ccg ccc tcc aca gcctcg gct cct gct gct gtg aac tcc tct gct 1152 Pro Pro Pro Ser Thr Ala SerAla Pro Ala Ala Val Asn Ser Ser Ala 370 375 380 tca gca gat aag cca ttatcc aac atg aag atc ctg act ctc ggg aag 1200 Ser Ala Asp Lys Pro Leu SerAsn Met Lys Ile Leu Thr Leu Gly Lys 385 390 395 400 ctg tcc cgg aac aaggat gaa gtg aag gcc atg att gag aaa ctc ggg 1248 Leu Ser Arg Asn Lys AspGlu Val Lys Ala Met Ile Glu Lys Leu Gly 405 410 415 ggg aag ttg acg gggacg gcc aac aag gct tcc ctg tgc atc agc acc 1296 Gly Lys Leu Thr Gly ThrAla Asn Lys Ala Ser Leu Cys Ile Ser Thr 420 425 430 aaa aag gag gtg gaaaag atg aat aag aag atg gag gaa gta aag gaa 1344 Lys Lys Glu Val Glu LysMet Asn Lys Lys Met Glu Glu Val Lys Glu 435 440 445 gcc aac atc cga gttgtg tct gag gac ttc ctc cag gac gtc tcc gcc 1392 Ala Asn Ile Arg Val ValSer Glu Asp Phe Leu Gln Asp Val Ser Ala 450 455 460 tcc acc aag agc cttcag gag ttg ttc tta gcg cac atc ttg tcc cct 1440 Ser Thr Lys Ser Leu GlnGlu Leu Phe Leu Ala His Ile Leu Ser Pro 465 470 475 480 tgg ggg gca gaggtg aag gca gag cct gtt gaa gtt gtg gcc cca aga 1488 Trp Gly Ala Glu ValLys Ala Glu Pro Val Glu Val Val Ala Pro Arg 485 490 495 ggg aag tca ggggct gcg ctc tcc aaa aaa agc aag ggc cag gtc aag 1536 Gly Lys Ser Gly AlaAla Leu Ser Lys Lys Ser Lys Gly Gln Val Lys 500 505 510 gag gaa ggt atcaac aaa tct gaa aag aga atg aaa tta act ctt aaa 1584 Glu Glu Gly Ile AsnLys Ser Glu Lys Arg Met Lys Leu Thr Leu Lys 515 520 525 gga gga gca gctgtg gat cct gat tct gga ctg gaa cac tct gcg cat 1632 Gly Gly Ala Ala ValAsp Pro Asp Ser Gly Leu Glu His Ser Ala His 530 535 540 gtc ctg gag aaaggt ggg aag gtc ttc agt gcc acc ctt ggc ctg gtg 1680 Val Leu Glu Lys GlyGly Lys Val Phe Ser Ala Thr Leu Gly Leu Val 545 550 555 560 gac atc gttaaa gga acc aac tcc tac tac aag ctg cag ctt ctg gag 1728 Asp Ile Val LysGly Thr Asn Ser Tyr Tyr Lys Leu Gln Leu Leu Glu 565 570 575 gac gac aaggaa aac agg tat tgg ata ttc agg tcc tgg ggc cgt gtg 1776 Asp Asp Lys GluAsn Arg Tyr Trp Ile Phe Arg Ser Trp Gly Arg Val 580 585 590 ggt acg gtgatc ggt agc aac aaa ctg gaa cag atg ccg tcc aag gag 1824 Gly Thr Val IleGly Ser Asn Lys Leu Glu Gln Met Pro Ser Lys Glu 595 600 605 gat gcc attgag cag ttc atg aaa tta tat gaa gaa aaa acc ggg aac 1872 Asp Ala Ile GluGln Phe Met Lys Leu Tyr Glu Glu Lys Thr Gly Asn 610 615 620 gct tgg cactcc aaa aat ttc acg aag tat ccc aaa aag ttt tac ccc 1920 Ala Trp His SerLys Asn Phe Thr Lys Tyr Pro Lys Lys Phe Tyr Pro 625 630 635 640 ctg gagatt gac tat ggc cag gat gaa gag gca gtg aag aag ctc aca 1968 Leu Glu IleAsp Tyr Gly Gln Asp Glu Glu Ala Val Lys Lys Leu Thr 645 650 655 gta aatcct ggc acc aag tcc aag ctc ccc aag cca gtt cag gac ctc 2016 Val Asn ProGly Thr Lys Ser Lys Leu Pro Lys Pro Val Gln Asp Leu 660 665 670 atc aagatg atc ttt gat gtg gaa agt atg aag aaa gcc atg gtg gag 2064 Ile Lys MetIle Phe Asp Val Glu Ser Met Lys Lys Ala Met Val Glu 675 680 685 tat gagatc gac ctt cag aag atg ccc ttg ggg aag ctg agc aaa agg 2112 Tyr Glu IleAsp Leu Gln Lys Met Pro Leu Gly Lys Leu Ser Lys Arg 690 695 700 cag atccag gcc gca tac tcc atc ctc agt gag gtc cag cag gcg gtg 2160 Gln Ile GlnAla Ala Tyr Ser Ile Leu Ser Glu Val Gln Gln Ala Val 705 710 715 720 tctcag ggc agc agc gac tct cag atc ctg gat ctc tca aat cgc ttt 2208 Ser GlnGly Ser Ser Asp Ser Gln Ile Leu Asp Leu Ser Asn Arg Phe 725 730 735 tacacc ctg atc ccc cac gac ttt ggg atg aag aag cct ccg ctc ctg 2256 Tyr ThrLeu Ile Pro His Asp Phe Gly Met Lys Lys Pro Pro Leu Leu 740 745 750 aacaat gca gac agt gtg cag gcc aag gtg gaa atg ctt gac aac ctg 2304 Asn AsnAla Asp Ser Val Gln Ala Lys Val Glu Met Leu Asp Asn Leu 755 760 765 ctggac atc gag gtg gcc tac agt ctg ctc agg gga ggg tct gat gat 2352 Leu AspIle Glu Val Ala Tyr Ser Leu Leu Arg Gly Gly Ser Asp Asp 770 775 780 agcagc aag gat ccc atc gat gtc aac tat gag aag ctc aaa act gac 2400 Ser SerLys Asp Pro Ile Asp Val Asn Tyr Glu Lys Leu Lys Thr Asp 785 790 795 800att aag gtg gtt gac aga gat tct gaa gaa gcc gag atc atc agg aag 2448 IleLys Val Val Asp Arg Asp Ser Glu Glu Ala Glu Ile Ile Arg Lys 805 810 815tat gtt aag aac act cat gca acc aca cac agt gcg tat gac ttg gaa 2496 TyrVal Lys Asn Thr His Ala Thr Thr His Ser Ala Tyr Asp Leu Glu 820 825 830gtc atc gat atc ttt aag ata gag cgt gaa ggc gaa tgc cag cgt tac 2544 ValIle Asp Ile Phe Lys Ile Glu Arg Glu Gly Glu Cys Gln Arg Tyr 835 840 845aag ccc ttt aag cag ctt cat aac cga aga ttg ctg tgg cac ggg tcc 2592 LysPro Phe Lys Gln Leu His Asn Arg Arg Leu Leu Trp His Gly Ser 850 855 860agg acc acc aac ttt gct ggg atc ctg tcc cag ggt ctt cgg ata gcc 2640 ArgThr Thr Asn Phe Ala Gly Ile Leu Ser Gln Gly Leu Arg Ile Ala 865 870 875880 ccg cct gaa gcg ccc gtg aca ggc tac atg ttt ggt aaa ggg atc tat 2688Pro Pro Glu Ala Pro Val Thr Gly Tyr Met Phe Gly Lys Gly Ile Tyr 885 890895 ttc gct gac atg gtc tcc aag agt gcc aac tac tac cat acg tct cag 2736Phe Ala Asp Met Val Ser Lys Ser Ala Asn Tyr Tyr His Thr Ser Gln 900 905910 gga gac cca ata ggc tta atc ctg ttg gga gaa gtt gcc ctt gga aac 2784Gly Asp Pro Ile Gly Leu Ile Leu Leu Gly Glu Val Ala Leu Gly Asn 915 920925 atg tat gaa ctg aag cac gct tca cat atc agc agg tta ccc aag ggc 2832Met Tyr Glu Leu Lys His Ala Ser His Ile Ser Arg Leu Pro Lys Gly 930 935940 aag cac agt gtc aaa ggt ttg ggc aaa act acc cct gat cct tca gct 2880Lys His Ser Val Lys Gly Leu Gly Lys Thr Thr Pro Asp Pro Ser Ala 945 950955 960 aac att agt ctg gat ggt gta gac gtt cct ctt ggg acc ggg att tca2928 Asn Ile Ser Leu Asp Gly Val Asp Val Pro Leu Gly Thr Gly Ile Ser 965970 975 tct ggt gtg ata gac acc tct cta cta tat aac gag tac att gtc tat2976 Ser Gly Val Ile Asp Thr Ser Leu Leu Tyr Asn Glu Tyr Ile Val Tyr 980985 990 gat att gct cag gta aat ctg aag tat ctg ctg aaa ctg aaa ttc aat3024 Asp Ile Ala Gln Val Asn Leu Lys Tyr Leu Leu Lys Leu Lys Phe Asn 9951000 1005 ttt aag acc tcc ctg tgg taa 3045 Phe Lys Thr Ser Leu Trp 101025 1014 PRT Homo sapiens 25 Met Ala Glu Ser Ser Asp Lys Leu Tyr Arg ValGlu Tyr Ala Lys Ser 1 5 10 15 Gly Arg Ala Ser Cys Lys Lys Cys Ser GluSer Ile Pro Lys Asp Ser 20 25 30 Leu Arg Met Ala Ile Met Val Gln Ser ProMet Phe Asp Gly Lys Val 35 40 45 Pro His Trp Tyr His Phe Ser Cys Phe TrpLys Val Gly His Ser Ile 50 55 60 Arg His Pro Asp Val Glu Val Asp Gly PheSer Glu Leu Arg Trp Asp 65 70 75 80 Asp Gln Gln Lys Val Lys Lys Thr AlaGlu Ala Gly Gly Val Thr Gly 85 90 95 Lys Gly Gln Asp Gly Ile Gly Ser LysAla Glu Lys Thr Leu Gly Asp 100 105 110 Phe Ala Ala Glu Tyr Ala Lys SerAsn Arg Ser Thr Cys Lys Gly Cys 115 120 125 Met Glu Lys Ile Glu Lys GlyGln Val Arg Leu Ser Lys Lys Met Val 130 135 140 Asp Pro Glu Lys Pro GlnLeu Gly Met Ile Asp Arg Trp Tyr His Pro 145 150 155 160 Gly Cys Phe ValLys Asn Arg Glu Glu Leu Gly Phe Arg Pro Glu Tyr 165 170 175 Ser Ala SerGln Leu Lys Gly Phe Ser Leu Leu Ala Thr Glu Asp Lys 180 185 190 Glu AlaLeu Lys Lys Gln Leu Pro Gly Val Lys Ser Glu Gly Lys Arg 195 200 205 LysGly Asp Glu Val Asp Gly Val Asp Glu Val Ala Lys Lys Lys Ser 210 215 220Lys Lys Glu Lys Asp Lys Asp Ser Lys Leu Glu Lys Ala Leu Lys Ala 225 230235 240 Gln Asn Asp Leu Ile Trp Asn Ile Lys Asp Glu Leu Lys Lys Val Cys245 250 255 Ser Thr Asn Asp Leu Lys Glu Leu Leu Ile Phe Asn Lys Gln GlnVal 260 265 270 Pro Ser Gly Glu Ser Ala Ile Leu Asp Arg Val Ala Asp GlyMet Val 275 280 285 Phe Gly Ala Leu Leu Pro Cys Glu Glu Cys Ser Gly GlnLeu Val Phe 290 295 300 Lys Ser Asp Ala Tyr Tyr Cys Thr Gly Asp Val ThrAla Trp Thr Lys 305 310 315 320 Cys Met Val Lys Thr Gln Thr Pro Asn ArgLys Glu Trp Val Thr Pro 325 330 335 Lys Glu Phe Arg Glu Ile Ser Tyr LeuLys Lys Leu Lys Val Lys Lys 340 345 350 Gln Asp Arg Ile Phe Pro Pro GluThr Ser Ala Ser Val Ala Ala Thr 355 360 365 Pro Pro Pro Ser Thr Ala SerAla Pro Ala Ala Val Asn Ser Ser Ala 370 375 380 Ser Ala Asp Lys Pro LeuSer Asn Met Lys Ile Leu Thr Leu Gly Lys 385 390 395 400 Leu Ser Arg AsnLys Asp Glu Val Lys Ala Met Ile Glu Lys Leu Gly 405 410 415 Gly Lys LeuThr Gly Thr Ala Asn Lys Ala Ser Leu Cys Ile Ser Thr 420 425 430 Lys LysGlu Val Glu Lys Met Asn Lys Lys Met Glu Glu Val Lys Glu 435 440 445 AlaAsn Ile Arg Val Val Ser Glu Asp Phe Leu Gln Asp Val Ser Ala 450 455 460Ser Thr Lys Ser Leu Gln Glu Leu Phe Leu Ala His Ile Leu Ser Pro 465 470475 480 Trp Gly Ala Glu Val Lys Ala Glu Pro Val Glu Val Val Ala Pro Arg485 490 495 Gly Lys Ser Gly Ala Ala Leu Ser Lys Lys Ser Lys Gly Gln ValLys 500 505 510 Glu Glu Gly Ile Asn Lys Ser Glu Lys Arg Met Lys Leu ThrLeu Lys 515 520 525 Gly Gly Ala Ala Val Asp Pro Asp Ser Gly Leu Glu HisSer Ala His 530 535 540 Val Leu Glu Lys Gly Gly Lys Val Phe Ser Ala ThrLeu Gly Leu Val 545 550 555 560 Asp Ile Val Lys Gly Thr Asn Ser Tyr TyrLys Leu Gln Leu Leu Glu 565 570 575 Asp Asp Lys Glu Asn Arg Tyr Trp IlePhe Arg Ser Trp Gly Arg Val 580 585 590 Gly Thr Val Ile Gly Ser Asn LysLeu Glu Gln Met Pro Ser Lys Glu 595 600 605 Asp Ala Ile Glu Gln Phe MetLys Leu Tyr Glu Glu Lys Thr Gly Asn 610 615 620 Ala Trp His Ser Lys AsnPhe Thr Lys Tyr Pro Lys Lys Phe Tyr Pro 625 630 635 640 Leu Glu Ile AspTyr Gly Gln Asp Glu Glu Ala Val Lys Lys Leu Thr 645 650 655 Val Asn ProGly Thr Lys Ser Lys Leu Pro Lys Pro Val Gln Asp Leu 660 665 670 Ile LysMet Ile Phe Asp Val Glu Ser Met Lys Lys Ala Met Val Glu 675 680 685 TyrGlu Ile Asp Leu Gln Lys Met Pro Leu Gly Lys Leu Ser Lys Arg 690 695 700Gln Ile Gln Ala Ala Tyr Ser Ile Leu Ser Glu Val Gln Gln Ala Val 705 710715 720 Ser Gln Gly Ser Ser Asp Ser Gln Ile Leu Asp Leu Ser Asn Arg Phe725 730 735 Tyr Thr Leu Ile Pro His Asp Phe Gly Met Lys Lys Pro Pro LeuLeu 740 745 750 Asn Asn Ala Asp Ser Val Gln Ala Lys Val Glu Met Leu AspAsn Leu 755 760 765 Leu Asp Ile Glu Val Ala Tyr Ser Leu Leu Arg Gly GlySer Asp Asp 770 775 780 Ser Ser Lys Asp Pro Ile Asp Val Asn Tyr Glu LysLeu Lys Thr Asp 785 790 795 800 Ile Lys Val Val Asp Arg Asp Ser Glu GluAla Glu Ile Ile Arg Lys 805 810 815 Tyr Val Lys Asn Thr His Ala Thr ThrHis Ser Ala Tyr Asp Leu Glu 820 825 830 Val Ile Asp Ile Phe Lys Ile GluArg Glu Gly Glu Cys Gln Arg Tyr 835 840 845 Lys Pro Phe Lys Gln Leu HisAsn Arg Arg Leu Leu Trp His Gly Ser 850 855 860 Arg Thr Thr Asn Phe AlaGly Ile Leu Ser Gln Gly Leu Arg Ile Ala 865 870 875 880 Pro Pro Glu AlaPro Val Thr Gly Tyr Met Phe Gly Lys Gly Ile Tyr 885 890 895 Phe Ala AspMet Val Ser Lys Ser Ala Asn Tyr Tyr His Thr Ser Gln 900 905 910 Gly AspPro Ile Gly Leu Ile Leu Leu Gly Glu Val Ala Leu Gly Asn 915 920 925 MetTyr Glu Leu Lys His Ala Ser His Ile Ser Arg Leu Pro Lys Gly 930 935 940Lys His Ser Val Lys Gly Leu Gly Lys Thr Thr Pro Asp Pro Ser Ala 945 950955 960 Asn Ile Ser Leu Asp Gly Val Asp Val Pro Leu Gly Thr Gly Ile Ser965 970 975 Ser Gly Val Ile Asp Thr Ser Leu Leu Tyr Asn Glu Tyr Ile ValTyr 980 985 990 Asp Ile Ala Gln Val Asn Leu Lys Tyr Leu Leu Lys Leu LysPhe Asn 995 1000 1005 Phe Lys Thr Ser Leu Trp 1010 26 39 DNA ArtificialSequence Description of Artificial Sequence Primer 26 cgtcgacccatggcggagtc ttcggataag ctctatcga 39 27 39 DNA Artificial SequenceDescription of Artificial Sequence Primer 27 ggaaacgcgt ttggtgccaggatttactgt cagcttctt 39 28 39 DNA Artificial Sequence Description ofArtificial Sequence Primer 28 ttgaaacgcg ttccagagtc acagctagat cttcgggta39 29 88 DNA Artificial Sequence Description of Artificial SequencePrimer 29 gtctcgaaag cggccgctta gcctccgaac tgtggatgcc tccacgcccacagctgaagg 60 aaattaaact gaacctttaa aaggtacc 88 30 17 DNA ArtificialSequence Description of Artificial Sequence Primer 30 tttgttcgcc cagactc17 31 22 DNA Artificial Sequence Description of Artificial SequencePrimer 31 tatgtttcag gttcaggggg ag 22 32 20 DNA Artificial SequenceDescription of Artificial Sequence Primer 32 gcggaagctg gaggagtgac 20 3320 DNA Artificial Sequence Description of Artificial Sequence Primer 33gtcactcctc cagcttccgc 20 34 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 34 aagccctgaa gaagcagctc 20 35 20 DNAArtificial Sequence Description of Artificial Sequence Primer 35gagctgcttc ttcagggctt 20 36 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 36 cagacaccca accggaagga 20 37 20 DNAArtificial Sequence Description of Artificial Sequence Primer 37tccttccggt tgggtgtctg 20 38 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 38 tccgcctcca ccaagagcct 20 39 20 DNAArtificial Sequence Description of Artificial Sequence Primer 39aggctcttgg tggaggcgga 20 40 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 40 tggcctggtg gacatcgtta 20 41 20 DNAArtificial Sequence Description of Artificial Sequence Primer 41taacgatgtc caccaggcca 20 42 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 42 gtattcttta ggcgagaggc 20 43 20 DNAArtificial Sequence Description of Artificial Sequence Primer 43tgacgaagtg ggcagaactg 20 44 21 DNA Artificial Sequence Description ofArtificial Sequence Primer 44 gagcaccccc tggaccagca c 21 45 20 DNAArtificial Sequence Description of Artificial Sequence Primer 45acagcgacta taccatgacc 20 46 3200 DNA Artificial Sequence Description ofArtificial SequencehPARP1/ hPARP2 Fusion 46 atgagaggct cccatcaccatcaccatcac gattacgata tcccaacgac cgaaaacctg 60 tattttcagg gcgccatggatccggaattc aaaggcctac gtcgacccat ggcggagtct 120 tcggataagc tctatcgagtcgagtacgcc aagagcgggc gcgcctcttg caagaaatgt 180 agcgagagca tccccaaggactcgctccgg atggccatca tggtgcagtc gcccatgttt 240 gatggaaaag tcccacactggtaccacttc tcctgcttct ggaaggtggg ccactccatc 300 cggcaccctg acgttgaggtggatgggttc tctgagcttc ggtgggatga ccagcagaaa 360 gtcaagaaga cagcggaagctggaggagtg acaggcaaag gccaggatgg aattggtagc 420 aaggcagaga agactctgggtgactttgca gcagagtatg tcaagtccaa cagaagtacg 480 tgcaaggggt gtatggagaagatagaaaag ggccaggtgc gcctgtccaa gaagatggtg 540 gacccggaga agccacagctaggcatgatt gaccgctggt accatccagg ctgctttgtc 600 aagaacaggg aggagctgggtttccggccc gagtacagtg cgagtcagct caagggcttc 660 agcctccttg ctacagaggataaagaagcc ctgaagaagc agctcccagg agtcaagagt 720 gaaggaaaga gtaaaggcgatgaggtggat ggagtggatg aagtggcgaa gaagaaatct 780 aaaaaagaaa aagacaaggatagtaagctt gaaaaagccc taaaggctca gaacgacctg 840 atctggaaca tcaaggacgagctaaagaaa gtgtgttcaa ctaatgacct gaaggagcta 900 ctcatcttca acaagcagcaagtgccttct ggggagtcgg cgatcttgga ccgagtagct 960 gatggcatgg tgttcggtgccctccttccc tgcgaggaat gctcgggtca gctggtcttc 1020 aagagcgatg cctattactgcactggggac gtcactgcct ggaccaagtg tatggtcaag 1080 acacagacac ccaaccggaaggagtgggta accccaaagg aattccgaga aatctcttac 1140 ctcaagaaat tgaaggttaaaaagcaggac cgtatattcc ccccagaaac cagcgcctcc 1200 gtggcggcca cgcctccgccctccacagcc tcggctcctg ctgctgtgaa ctcctctgct 1260 tcagcagata agccattatccaacatgaag atcctgactc tcgggaagct gtcccggaac 1320 aaggatgaag tgaaggccatgattgagaaa ctcgggggga agttgacggg gacggccaac 1380 aaggcttccc tgtgcatcagcaccaaaaag gaggtggaaa agatgaataa gaagatggag 1440 gaagtaaagg aagccaacatccgagttgtg tctgaggact tcctccagga cgtctccgcc 1500 tccaccaaga gccttcaggagttgttctta gcgcacatct tgtccccttg gggggcagag 1560 gtgaaggcag agcctgttgaagttgtggcc ccaagaggga agtcaggggc tgcgctctcc 1620 aaaaaaagca agggccaggtcaaggaggaa ggtatcaaca aatctgaaaa gagaatgaaa 1680 ttaactctta aaggaggagcagctgtggat cctgattctg gactggaaca ctctgcgcat 1740 gtcctggaga aaggtgggaaggtcttcagt gccacccttg gcctggtgga catcgttaaa 1800 ggaaccaact cctactacaagctgcagctt ctggaggacg acaaggaaaa caggtattgg 1860 atattcaggt cctggggccgtgtgggtacg gtgatcggta gcaacaaact ggaacagatg 1920 ccgtccaagg aggatgccattgagcacttc atgaaattat atgaagaaaa aaccgggaac 1980 gcttggcact ccaaaaatttcacgaagtat cccaaaaagt tctaccccct ggagattgac 2040 tatggccagg atgaagaggcagtgaagaag ctgacagtaa atcctggcac caaacgcgtt 2100 ccagagtcac agctagatcttcgggtacag gagttaataa agttgatctg taatgttcag 2160 gccatggaag aaatgatgatggaaatgaag tataatacca agaaagcccc tcttgggaag 2220 ctgacagtgg cgcaaatcaaggcaggttac cagtctctta agaagattga ggattgtatt 2280 cgggctggcc agcatggacgagctctcatg gaagcatgca atgaattcta caccaggatt 2340 ccgcatgact ttggactccgtactcctcca ctaatccgga cacagaagga actgtcagaa 2400 aaaatacaat tactagaggctttgggagac attgaaattg ctattaagct ggtgaaaaca 2460 gagctacaaa gcccagaacacccattggac caacactata gaaacctaca ttgtgccttg 2520 cgcccccttg accatgaaagttacgagttc aaagtgattt cccagtacct acaatctacc 2580 catgctccca cacacagcgactataccatg accttgctgg atttgtttga agtggagaag 2640 gatggtgaga aagaagccttcagagaggac cttcataaca ggatgcttct atggcatggt 2700 tccaggatga gtaactgggtgggaatcttg agccatgggc ttcgaattgc cccacctgaa 2760 gctcccatca caggttacatgtttgggaaa ggaatctact ttgctgacat gtcttccaag 2820 agtgccaatt actgctttgcctctcgccta aagaatacag gactgctgct cttatcagag 2880 gtagctctag gtcagtgtaatgaactacta gaggccaatc ctaaggccga aggattgctt 2940 caaggtaaac atagcaccaaggggctgggc aagatggctc ccagttctgc ccacttcgtc 3000 accctgaatg ggagtacagtgccattagga ccagcaagtg acacaggaat tctgaatcca 3060 gatggttata ccctcaactacaatgaatat attgtatata accccaacca ggtccgtatg 3120 cggtaccttt taaaggttcagtttaatttc cttcagctgt gggcgtggag gcatccacag 3180 ttcggaggct aagcggccgc3200 47 1063 PRT Artificial Sequence Description of ArtificialSequencehPARP1/ hPARP2 Fusion 47 Met Arg Gly Ser His His His His His HisAsp Tyr Asp Ile Pro Thr 1 5 10 15 Thr Glu Asn Leu Tyr Phe Gln Gly AlaMet Asp Pro Glu Phe Lys Gly 20 25 30 Leu Arg Arg Pro Met Ala Glu Ser SerAsp Lys Leu Tyr Arg Val Glu 35 40 45 Tyr Ala Lys Ser Gly Arg Ala Ser CysLys Lys Cys Ser Glu Ser Ile 50 55 60 Pro Lys Asp Ser Leu Arg Met Ala IleMet Val Gln Ser Pro Met Phe 65 70 75 80 Asp Gly Lys Val Pro His Trp TyrHis Phe Ser Cys Phe Trp Lys Val 85 90 95 Gly His Ser Ile Arg His Pro AspVal Glu Val Asp Gly Phe Ser Glu 100 105 110 Leu Arg Trp Asp Asp Gln GlnLys Val Lys Lys Thr Ala Glu Ala Gly 115 120 125 Gly Val Thr Gly Lys GlyGln Asp Gly Ile Gly Ser Lys Ala Glu Lys 130 135 140 Thr Leu Gly Asp PheAla Ala Glu Tyr Val Lys Ser Asn Arg Ser Thr 145 150 155 160 Cys Lys GlyCys Met Glu Lys Ile Glu Lys Gly Gln Val Arg Leu Ser 165 170 175 Lys LysMet Val Asp Pro Glu Lys Pro Gln Leu Gly Met Ile Asp Arg 180 185 190 TrpTyr His Pro Gly Cys Phe Val Lys Asn Arg Glu Glu Leu Gly Phe 195 200 205Arg Pro Glu Tyr Ser Ala Ser Gln Leu Lys Gly Phe Ser Leu Leu Ala 210 215220 Thr Glu Asp Lys Glu Ala Leu Lys Lys Gln Leu Pro Gly Val Lys Ser 225230 235 240 Glu Gly Lys Ser Lys Gly Asp Glu Val Asp Gly Val Asp Glu ValAla 245 250 255 Lys Lys Lys Ser Lys Lys Glu Lys Asp Lys Asp Ser Lys LeuGlu Lys 260 265 270 Ala Leu Lys Ala Gln Asn Asp Leu Ile Trp Asn Ile LysAsp Glu Leu 275 280 285 Lys Lys Val Cys Ser Thr Asn Asp Leu Lys Glu LeuLeu Ile Phe Asn 290 295 300 Lys Gln Gln Val Pro Ser Gly Glu Ser Ala IleLeu Asp Arg Val Ala 305 310 315 320 Asp Gly Met Val Phe Gly Ala Leu LeuPro Cys Glu Glu Cys Ser Gly 325 330 335 Gln Leu Val Phe Lys Ser Asp AlaTyr Tyr Cys Thr Gly Asp Val Thr 340 345 350 Ala Trp Thr Lys Cys Met ValLys Thr Gln Thr Pro Asn Arg Lys Glu 355 360 365 Trp Val Thr Pro Lys GluPhe Arg Glu Ile Ser Tyr Leu Lys Lys Leu 370 375 380 Lys Val Lys Lys GlnAsp Arg Ile Phe Pro Pro Glu Thr Ser Ala Ser 385 390 395 400 Val Ala AlaThr Pro Pro Pro Ser Thr Ala Ser Ala Pro Ala Ala Val 405 410 415 Asn SerSer Ala Ser Ala Asp Lys Pro Leu Ser Asn Met Lys Ile Leu 420 425 430 ThrLeu Gly Lys Leu Ser Arg Asn Lys Asp Glu Val Lys Ala Met Ile 435 440 445Glu Lys Leu Gly Gly Lys Leu Thr Gly Thr Ala Asn Lys Ala Ser Leu 450 455460 Cys Ile Ser Thr Lys Lys Glu Val Glu Lys Met Asn Lys Lys Met Glu 465470 475 480 Glu Val Lys Glu Ala Asn Ile Arg Val Val Ser Glu Asp Phe LeuGln 485 490 495 Asp Val Ser Ala Ser Thr Lys Ser Leu Gln Glu Leu Phe LeuAla His 500 505 510 Ile Leu Ser Pro Trp Gly Ala Glu Val Lys Ala Glu ProVal Glu Val 515 520 525 Val Ala Pro Arg Gly Lys Ser Gly Ala Ala Leu SerLys Lys Ser Lys 530 535 540 Gly Gln Val Lys Glu Glu Gly Ile Asn Lys SerGlu Lys Arg Met Lys 545 550 555 560 Leu Thr Leu Lys Gly Gly Ala Ala ValAsp Pro Asp Ser Gly Leu Glu 565 570 575 His Ser Ala His Val Leu Glu LysGly Gly Lys Val Phe Ser Ala Thr 580 585 590 Leu Gly Leu Val Asp Ile ValLys Gly Thr Asn Ser Tyr Tyr Lys Leu 595 600 605 Gln Leu Leu Glu Asp AspLys Glu Asn Arg Tyr Trp Ile Phe Arg Ser 610 615 620 Trp Gly Arg Val GlyThr Val Ile Gly Ser Asn Lys Leu Glu Gln Met 625 630 635 640 Pro Ser LysGlu Asp Ala Ile Glu His Phe Met Lys Leu Tyr Glu Glu 645 650 655 Lys ThrGly Asn Ala Trp His Ser Lys Asn Phe Thr Lys Tyr Pro Lys 660 665 670 LysPhe Tyr Pro Leu Glu Ile Asp Tyr Gly Gln Asp Glu Glu Ala Val 675 680 685Lys Lys Leu Thr Val Asn Pro Gly Thr Lys Arg Val Pro Glu Ser Gln 690 695700 Leu Asp Leu Arg Val Gln Glu Leu Ile Lys Leu Ile Cys Asn Val Gln 705710 715 720 Ala Met Glu Glu Met Met Met Glu Met Lys Tyr Asn Thr Lys LysAla 725 730 735 Pro Leu Gly Lys Leu Thr Val Ala Gln Ile Lys Ala Gly TyrGln Ser 740 745 750 Leu Lys Lys Ile Glu Asp Cys Ile Arg Ala Gly Gln HisGly Arg Ala 755 760 765 Leu Met Glu Ala Cys Asn Glu Phe Tyr Thr Arg IlePro His Asp Phe 770 775 780 Gly Leu Arg Thr Pro Pro Leu Ile Arg Thr GlnLys Glu Leu Ser Glu 785 790 795 800 Lys Ile Gln Leu Leu Glu Ala Leu GlyAsp Ile Glu Ile Ala Ile Lys 805 810 815 Leu Val Lys Thr Glu Leu Gln SerPro Glu His Pro Leu Asp Gln His 820 825 830 Tyr Arg Asn Leu His Cys AlaLeu Arg Pro Leu Asp His Glu Ser Tyr 835 840 845 Glu Phe Lys Val Ile SerGln Tyr Leu Gln Ser Thr His Ala Pro Thr 850 855 860 His Ser Asp Tyr ThrMet Thr Leu Leu Asp Leu Phe Glu Val Glu Lys 865 870 875 880 Asp Gly GluLys Glu Ala Phe Arg Glu Asp Leu His Asn Arg Met Leu 885 890 895 Leu TrpHis Gly Ser Arg Met Ser Asn Trp Val Gly Ile Leu Ser His 900 905 910 GlyLeu Arg Ile Ala Pro Pro Glu Ala Pro Ile Thr Gly Tyr Met Phe 915 920 925Gly Lys Gly Ile Tyr Phe Ala Asp Met Ser Ser Lys Ser Ala Asn Tyr 930 935940 Cys Phe Ala Ser Arg Leu Lys Asn Thr Gly Leu Leu Leu Leu Ser Glu 945950 955 960 Val Ala Leu Gly Gln Cys Asn Glu Leu Leu Glu Ala Asn Pro LysAla 965 970 975 Glu Gly Leu Leu Gln Gly Lys His Ser Thr Lys Gly Leu GlyLys Met 980 985 990 Ala Pro Ser Ser Ala His Phe Val Thr Leu Asn Gly SerThr Val Pro 995 1000 1005 Leu Gly Pro Ala Ser Asp Thr Gly Ile Leu AsnPro Asp Gly Tyr Thr 1010 1015 1020 Leu Asn Tyr Asn Glu Tyr Ile Val TyrAsn Pro Asn Gln Val Arg Met 1025 1030 1035 1040 Arg Tyr Leu Leu Lys ValGln Phe Asn Phe Leu Gln Leu Trp Ala Trp 1045 1050 1055 Arg His Pro GlnPhe Gly Gly 1060 48 39 DNA Artificial Sequence Description of ArtificialSequencePrimer 48 cgtcgaccca tggcggcgcg gcggcgacgg agcaccggc 39 49 39DNA Artificial Sequence Description of Artificial Sequence Primer 49tggaacgcgt ttcaagggag atttaagaga ttcctcttt 39 50 20 DNA ArtificialSequence Description of Artificial Sequence Primer 50 ccaggtccgtatgcggtacc 20 51 39 DNA Artificial Sequence Description of ArtificialSequence Primer 51 gccacgatgg gtaccgcggc cgctcaccac agctgaagg 39 52 20DNA Artificial Sequence Description of Artificial Sequence Primer 52ggtgacgaag tgggcagaac 20 53 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 53 ttctgcccac ttcgtcaccc 20 54 20 DNAArtificial Sequence Description of Artificial Sequence Primer 54cgcaaggcac aatgtaggtt 20 55 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 55 gcctctcgcc taaagaatac 20 56 20 DNAArtificial Sequence Description of Artificial Sequence Primer 56aagcaatcct tcggccttag 20 57 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 57 agttctgccc acttcgtcac 20 58 1874 DNAArtificial Sequence Description of Artificial SequencehPARP2 + His Tag58 atgagaggct cccatcacca tcaccatcac gattacgata tcccaacgac cgaaaacctg 60tattttcagg gcgccatgga tccggaattc aaaggcctac gtcgacccat ggcggcgcgg 120cggcgacgga gcaccggcgg cggcagggcg agagcattaa atgaaagcaa aagagttaat 180aatggcaaca cggctccaga agactcttcc cctgccaaga aaactcgtag atgccagaga 240caggagtcga aaaagatgcc tgtggctgga ggaaaagcta ataaggacag gacagaagac 300aagcaagatg gtatgccagg aaggtcatgg gccagcaaaa gggtctccga atctgtgaag 360gccttgctgt taaagggcaa agctcctgtg gacccagagt gtacagccaa ggtggggaag 420gctcatgtgt attgtgaagg aaatgatgtc tatgatgtca tgctaaatca gaccaatctc 480cagttcaaca acaacaagta ctatctgatt cagctattag aagatgatgc ccagaggaac 540ttcagtgttt ggatgagatg gggccgagtt gggaaaatgg gacagcacag cctggtggct 600tgttcaggca atctcaacaa ggccaaggaa atctttcaga agaaattcct tgacaaaacg 660aaaaacaatt gggaagatcg agaaaagttt gagaaggtgc ctggaaaata tgatatgcta 720cagatggact atgccaccaa tactcaggat gaagaggaaa caaagaaaga ggaatctctt 780aaatctccct tgaagccaga gtcacagcta gatcttcggg tacaggagtt aataaagttg 840atctgtaatg ttcaggccat ggaagaaatg atgatggaaa tgaagtataa taccaagaaa 900gcccctcttg ggaagctgac agtggcgcaa atcaaggcag gttaccagtc tcttaagaag 960attgaggatt gtattcgggc tggccagcat ggacgagctc tcatggaagc atgcaatgaa 1020ttctacacca ggattccgca tgactttgga ctccgtactc ctccactaat ccggacacag 1080aaggaactgt cagaaaaaat acaattacta gaggctttgg gagacattga aattgctatt 1140aagctggtga aaacagagct acaaagccca gaacacccat tggaccaaca ctatagaaac 1200ctacattgtg ccttgcgccc ccttgaccat gaaagttacg agttcaaagt gatttcccag 1260tacctacaat ctacccatgc tcccacacac agcgactata ccatgacctt gctggatttg 1320tttgaagtgg agaaggatgg tgagaaagaa gccttcagag aggaccttca taacaggatg 1380cttctatggc atggttccag gatgagtaac tgggtgggaa tcttgagcca tgggcttcga 1440attgccccac ctgaagctcc catcacaggt tacatgtttg ggaaaggaat ctactttgct 1500gacatgtctt ccaagagtgc caattactgc tttgcctctc gcctaaagaa tacaggactg 1560ctgctcttat cagaggtagc tctaggtcag tgtaatgaac tactagaggc caatcctaag 1620gccgaaggat tgcttcaagg taaacatagc accaaggggc tgggcaagat ggctcccagt 1680tctgcccact tcgtcaccct gaatgggagt acagtgccat taggaccagc aagtgacaca 1740ggaattctga atccagatgg ttataccctc aactacaatg aatatattgt atataacccc 1800aaccaggtcc gtatgcggta ccttttaaag gttcagttta atttccttca gctgtggtga 1860gcggccgcgg tacc 1874 59 619 PRT Artificial Sequence Description ofArtificial SequencehPARP2 + His Tag 59 Met Arg Gly Ser His His His HisHis His Asp Tyr Asp Ile Pro Thr 1 5 10 15 Thr Glu Asn Leu Tyr Phe GlnGly Ala Met Asp Pro Glu Phe Lys Gly 20 25 30 Leu Arg Arg Pro Met Ala AlaArg Arg Arg Arg Ser Thr Gly Gly Gly 35 40 45 Arg Ala Arg Ala Leu Asn GluSer Lys Arg Val Asn Asn Gly Asn Thr 50 55 60 Ala Pro Glu Asp Ser Ser ProAla Lys Lys Thr Arg Arg Cys Gln Arg 65 70 75 80 Gln Glu Ser Lys Lys MetPro Val Ala Gly Gly Lys Ala Asn Lys Asp 85 90 95 Arg Thr Glu Asp Lys GlnAsp Gly Met Pro Gly Arg Ser Trp Ala Ser 100 105 110 Lys Arg Val Ser GluSer Val Lys Ala Leu Leu Leu Lys Gly Lys Ala 115 120 125 Pro Val Asp ProGlu Cys Thr Ala Lys Val Gly Lys Ala His Val Tyr 130 135 140 Cys Glu GlyAsn Asp Val Tyr Asp Val Met Leu Asn Gln Thr Asn Leu 145 150 155 160 GlnPhe Asn Asn Asn Lys Tyr Tyr Leu Ile Gln Leu Leu Glu Asp Asp 165 170 175Ala Gln Arg Asn Phe Ser Val Trp Met Arg Trp Gly Arg Val Gly Lys 180 185190 Met Gly Gln His Ser Leu Val Ala Cys Ser Gly Asn Leu Asn Lys Ala 195200 205 Lys Glu Ile Phe Gln Lys Lys Phe Leu Asp Lys Thr Lys Asn Asn Trp210 215 220 Glu Asp Arg Glu Lys Phe Glu Lys Val Pro Gly Lys Tyr Asp MetLeu 225 230 235 240 Gln Met Asp Tyr Ala Thr Asn Thr Gln Asp Glu Glu GluThr Lys Lys 245 250 255 Glu Glu Ser Leu Lys Ser Pro Leu Lys Pro Glu SerGln Leu Asp Leu 260 265 270 Arg Val Gln Glu Leu Ile Lys Leu Ile Cys AsnVal Gln Ala Met Glu 275 280 285 Glu Met Met Met Glu Met Lys Tyr Asn ThrLys Lys Ala Pro Leu Gly 290 295 300 Lys Leu Thr Val Ala Gln Ile Lys AlaGly Tyr Gln Ser Leu Lys Lys 305 310 315 320 Ile Glu Asp Cys Ile Arg AlaGly Gln His Gly Arg Ala Leu Met Glu 325 330 335 Ala Cys Asn Glu Phe TyrThr Arg Ile Pro His Asp Phe Gly Leu Arg 340 345 350 Thr Pro Pro Leu IleArg Thr Gln Lys Glu Leu Ser Glu Lys Ile Gln 355 360 365 Leu Leu Glu AlaLeu Gly Asp Ile Glu Ile Ala Ile Lys Leu Val Lys 370 375 380 Thr Glu LeuGln Ser Pro Glu His Pro Leu Asp Gln His Tyr Arg Asn 385 390 395 400 LeuHis Cys Ala Leu Arg Pro Leu Asp His Glu Ser Tyr Glu Phe Lys 405 410 415Val Ile Ser Gln Tyr Leu Gln Ser Thr His Ala Pro Thr His Ser Asp 420 425430 Tyr Thr Met Thr Leu Leu Asp Leu Phe Glu Val Glu Lys Asp Gly Glu 435440 445 Lys Glu Ala Phe Arg Glu Asp Leu His Asn Arg Met Leu Leu Trp His450 455 460 Gly Ser Arg Met Ser Asn Trp Val Gly Ile Leu Ser His Gly LeuArg 465 470 475 480 Ile Ala Pro Pro Glu Ala Pro Ile Thr Gly Tyr Met PheGly Lys Gly 485 490 495 Ile Tyr Phe Ala Asp Met Ser Ser Lys Ser Ala AsnTyr Cys Phe Ala 500 505 510 Ser Arg Leu Lys Asn Thr Gly Leu Leu Leu LeuSer Glu Val Ala Leu 515 520 525 Gly Gln Cys Asn Glu Leu Leu Glu Ala AsnPro Lys Ala Glu Gly Leu 530 535 540 Leu Gln Gly Lys His Ser Thr Lys GlyLeu Gly Lys Met Ala Pro Ser 545 550 555 560 Ser Ala His Phe Val Thr LeuAsn Gly Ser Thr Val Pro Leu Gly Pro 565 570 575 Ala Ser Asp Thr Gly IleLeu Asn Pro Asp Gly Tyr Thr Leu Asn Tyr 580 585 590 Asn Glu Tyr Ile ValTyr Asn Pro Asn Gln Val Arg Met Arg Tyr Leu 595 600 605 Leu Lys Val GlnPhe Asn Phe Leu Gln Leu Trp 610 615 60 20 DNA Artificial SequenceDescription of Artificial Sequence Primer 60 ggagacattg aaattgctat 20 6121 DNA Artificial Sequence Description of Artificial Sequence Primer 61gaacacccat tggaccaaca c 21 62 21 DNA Artificial Sequence Description ofArtificial Sequence Primer 62 gaggtatata ttaatgtatc g 21 63 18 DNAArtificial Sequence Description of Artificial Sequence Primer 63gatttaatct gtatcagg 18 64 738 DNA Artificial Sequence Description ofArtificial SequencehPARP2 Fragment + C-Terminal His Tag 64 ggagacattgaaattgctat taagctggtg aaaacagagc tacaaagccc agaacaccca 60 ttggaccaacactatagaaa cctacattgt gccttgcgcc cccttgacca tgaaagttac 120 gagttcaaagtgatttccca gtacctacaa tctacccatg ctcccacaca cagcgactat 180 accatgaccttgctggattt gtttgaagtg gagaaggatg gtgagaaaga agccttcaga 240 gaggaccttcataacaggat gcttctatgg catggttcca ggatgagtaa ctgggtggga 300 atcttgagccatgggcttcg aattgcccca cctgaagctc ccatcacagg ttacatgttt 360 gggaaaggaatctactttgc tgacatgtct tccaagagtg ccaattactg ctttgcctct 420 cgcctaaagaatacaggact gctgctctta tcagaggtag ctctaggtca gtgtaatgaa 480 ctactagaggccaatcctaa ggccgaagga ttgcttcaag gtaaacatag caccaagggg 540 ctgggcaagatggctcccag ttctgcccac ttcgtcaccc tgaatgggag tacagtgcca 600 ttaggaccagcaagtgacac aggaattctg aatccagatg gttataccct caactacaat 660 gaatatattgtatataaccc caaccaggtc cgtatgcggt accttttaaa ggttcagttt 720 aatttccttcagctgtgg 738 65 275 PRT Artificial Sequence Description of ArtificialSequencehPARP2 Fragment + C-Terminal His Tag 65 Gly Asp Ile Glu Ile AlaIle Lys Leu Val Lys Thr Glu Leu Gln Ser 1 5 10 15 Pro Glu His Pro LeuAsp Gln His Tyr Arg Asn Leu His Cys Ala Leu 20 25 30 Arg Pro Leu Asp HisGlu Ser Tyr Glu Phe Lys Val Ile Ser Gln Tyr 35 40 45 Leu Gln Ser Thr HisAla Pro Thr His Ser Asp Tyr Thr Met Thr Leu 50 55 60 Leu Asp Leu Phe GluVal Glu Lys Asp Gly Glu Lys Glu Ala Phe Arg 65 70 75 80 Glu Asp Leu HisAsn Arg Met Leu Leu Trp His Gly Ser Arg Met Ser 85 90 95 Asn Trp Val GlyIle Leu Ser His Gly Leu Arg Ile Ala Pro Pro Glu 100 105 110 Ala Pro IleThr Gly Tyr Met Phe Gly Lys Gly Ile Tyr Phe Ala Asp 115 120 125 Met SerSer Lys Ser Ala Asn Tyr Cys Phe Ala Ser Arg Leu Lys Asn 130 135 140 ThrGly Leu Leu Leu Leu Ser Glu Val Ala Leu Gly Gln Cys Asn Glu 145 150 155160 Leu Leu Glu Ala Asn Pro Lys Ala Glu Gly Leu Leu Gln Gly Lys His 165170 175 Ser Thr Lys Gly Leu Gly Lys Met Ala Pro Ser Ser Ala His Phe Val180 185 190 Thr Leu Asn Gly Ser Thr Val Pro Leu Gly Pro Ala Ser Asp ThrGly 195 200 205 Ile Leu Asn Pro Asp Gly Tyr Thr Leu Asn Tyr Asn Glu TyrIle Val 210 215 220 Tyr Asn Pro Asn Gln Val Arg Met Arg Tyr Leu Leu LysVal Gln Phe 225 230 235 240 Asn Phe Leu Gln Leu Trp Lys Gly Glu Phe GluAla Tyr Val Glu Gln 245 250 255 Lys Leu Ile Ser Glu Glu Asp Leu Asn SerAla Val Asp His His His 260 265 270 His His His 275 66 687 DNAArtificial Sequence Description of Artificial SequencehPARP2 Fragment +C-Terminal His Tag 66 gaacacccat tggaccaaca ctatagaaac ctacattgtgccttgcgccc ccttgaccat 60 gaaagttacg agttcaaagt gatttcccag tacctacaatctacccatgc tcccacacac 120 agcgactata ccatgacctt gctggatttg tttgaagtggagaaggatgg tgagaaagaa 180 gccttcagag aggaccttca taacaggatg cttctatggcatggttccag gatgagtaac 240 tgggtgggaa tcttgagcca tgggcttcga attgccccacctgaagctcc catcacaggt 300 tacatgtttg ggaaaggaat ctactttgct gacatgtcttccaagagtgc caattactgc 360 tttgcctctc gcctaaagaa tacaggactg ctgctcttatcagaggtagc tctaggtcag 420 tgtaatgaac tactagaggc caatcctaag gccgaaggattgcttcaagg taaacatagc 480 accaaggggc tgggcaagat ggctcccagt tctgcccacttcgtcaccct gaatgggagt 540 acagtgccat taggaccagc aagtgacaca ggaattctgaatccagatgg ttataccctc 600 aactacaatg aatatattgt atataacccc aaccaggtccgtatgcggta ccttttaaag 660 gttcagttta atttccttca gctgtgg 687 67 258 PRTArtificial Sequence Description of Artificial SequencehPARP2 Fragment +C-Terminal His Tag 67 Glu His Pro Leu Asp Gln His Tyr Arg Asn Leu HisCys Ala Leu Arg 1 5 10 15 Pro Leu Asp His Glu Ser Tyr Glu Phe Lys ValIle Ser Gln Tyr Leu 20 25 30 Gln Ser Thr His Ala Pro Thr His Ser Asp TyrThr Met Thr Leu Leu 35 40 45 Asp Leu Phe Glu Val Glu Lys Asp Gly Glu LysGlu Ala Phe Arg Glu 50 55 60 Asp Leu His Asn Arg Met Leu Leu Trp His GlySer Arg Met Ser Asn 65 70 75 80 Trp Val Gly Ile Leu Ser His Gly Leu ArgIle Ala Pro Pro Glu Ala 85 90 95 Pro Ile Thr Gly Tyr Met Phe Gly Lys GlyIle Tyr Phe Ala Asp Met 100 105 110 Ser Ser Lys Ser Ala Asn Tyr Cys PheAla Ser Arg Leu Lys Asn Thr 115 120 125 Gly Leu Leu Leu Leu Ser Glu ValAla Leu Gly Gln Cys Asn Glu Leu 130 135 140 Leu Glu Ala Asn Pro Lys AlaGlu Gly Leu Leu Gln Gly Lys His Ser 145 150 155 160 Thr Lys Gly Leu GlyLys Met Ala Pro Ser Ser Ala His Phe Val Thr 165 170 175 Leu Asn Gly SerThr Val Pro Leu Gly Pro Ala Ser Asp Thr Gly Ile 180 185 190 Leu Asn ProAsp Gly Tyr Thr Leu Asn Tyr Asn Glu Tyr Ile Val Tyr 195 200 205 Asn ProAsn Gln Val Arg Met Arg Tyr Leu Leu Lys Val Gln Phe Asn 210 215 220 PheLeu Gln Leu Trp Lys Gly Glu Phe Glu Ala Tyr Val Glu Gln Lys 225 230 235240 Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His His His His 245250 255 His His 68 7 PRT artificial sequence Common catalytic motif forhPARP proteins 68 Gly Xaa Xaa Xaa Gly Lys Gly 1 5

What is claimed is:
 1. A purified and isolated hPARP2 polypeptide,comprising the amino acid sequence defined in SEQ ID NO:2 or afunctional derivative thereof.
 2. A polynucleotide encoding the hPARP2polypeptide according to claim
 1. 3. The polynucleotide according toclaim 2, comprising the nucleotide sequence defined in SEQ ID NO:1.
 4. Apolynucleotide encoding a hPARP2 polypeptide selected from the groupconsisting of: a) the polynucleotide according to claim 2, b) thepolynucleotide according to claim 3, and c) a polynucleotide thathybridizes under moderately stringent hybridization conditions to thecomplement of the polynucleotide of (a) or (b).
 5. An hPARP2 polypeptideencoded by the polynucleotide according to claim
 4. 6. Thepolynucleotide according to claim 4, which is a DNA molecule or an RNAmolecule.
 7. The polynucleotide according to claim 6, which furthercomprises a detectable label moiety.
 8. An expression construct,comprising the polynucleotide according to claim
 4. 9. A host celltransformed or transfected with the expression construct according toclaim
 8. 10. The polynucleotide according to claim 4, wherein thepolynucleotide is operatively linked to a heterologous promoter.
 11. Ahost cell, comprising the polynucleotide according to claim
 10. 12. Amethod for producing a polypeptide having an amino acid sequence definedby SEQ ID NO:2, comprising the steps of: a) growing the host cellaccording to claim 9 or 11 under conditions appropriate for expressionof the polypeptide; and b) isolating the polypeptide from the host cellor the medium in which the host cell is grown.
 13. An antibody that isspecifically immunoreactive with the polypeptide according to claim 1.14. The antibody according to claim 13, wherein the antibody is selectedfrom the group consisting of monoclonal antibodies, polyclonalantibodies, single chain antibodies (scFv antibodies), chimericantibodies, multifunctional/multispecific antibodies, humanizedantibodies, human antibodies, CDR-grafted antibodies, Fab fragments,Fab′ fragments, F(ab′)₂ fragments, Fv fragments, diabodies; linearantibodies; single-chain antibody molecules; and multispecificantibodies formed from antibody fragments.
 15. A cell line that producesthe antibody according to claim
 13. 16. An anti-idiotype antibody thatis specifically immunoreactive with an antibody according to claim 14.17. A method for identifying a specific binding partner of the hPARP2polypeptide according to claim 1, comprising: a) contacting the hPARP2polypeptide with a test compound under conditions that permit binding ofthe hPARP2 polypeptide and the test compound; b) detecting binding ofthe test compound and the hPARP2 polypeptide; and c) identifying thetest compound as a specific binding partner of the hPARP2 polypeptide.18. The method according to claim 17, wherein said specific bindingpartner modulates a biological activity of the hPARP2 polypeptide. 19.The method according to claim 18, wherein said specific binding partnerinhibits a biological activity of the hPARP2 polypeptide.
 20. The methodaccording to claim 18, wherein said specific binding partner enhances abiological activity of the hPARP2 polypeptide.
 21. A method foridentifying a specific binding partner of the hparp2 polynucleotideaccording to claim 2, comprising: a) contacting the hparp2polynucleotide with a test compound under conditions that permit bindingof the hparp2 polynucleotide and the test compound; b) detecting bindingof the test compound and the hparp2 polynucleotide; and c) identifyingthe test compound as a specific binding partner of the hparp2polynucleotide.
 22. The method according to claim 21, wherein saidspecific binding partner modulates expression of the hPARP2 polypeptide.23. The method according to claim 22, wherein said specific bindingpartner inhibits expression of the hPARP2 polypeptide.
 24. A methodaccording to claim 22, wherein said specific binding partner enhancesexpression of the hPARP2 polypeptide.
 25. A method of treating a humansubject having a disorder mediated by poly(ADP-ribose) polymeraseactivity, comprising administering to said subject an hPARP2 antagonistin an amount effective for inhibiting expression or activity of thepolypeptide according to claim
 1. 26. The method according to claim 25,wherein the hPARP1 antagonist inhibits hPARP1.
 27. The method accordingto claim 25, wherein said disorder is selected from the group consistingof inflammatory disorders, neurological disorders, cardiovasculardisorders, and disorders of neoplastic tissue growth.
 28. The methodaccording to claim 27, wherein said disorder is an inflammatorydisorder.
 29. The method according to claim 25, wherein said disorder ischaracterized by reperfusion injury.
 30. The method according to claim29, wherein said disorder is selected from the group consisting ofischemic stroke, hemorrhagic shock, myocardial ischemia or infarction,transplantation, and cerebral vasospasm.
 31. The method according toclaim 28, wherein said disorder is selected from the group consisting ofrheumatoid arthritis, osteoarthritis, gouty arthritis, spondylitis;Behcet disease; sepsis, septic shock, endotoxic shock, gram negativesepsis, gram positive sepsis, toxic shock syndrome; multiple organinjury syndrome secondary to septicemia, trauma, or hemorrhage; allergicconjunctivitis, vernal conjunctivitis, uveitis, thyroid-associatedophthalmopathy; cosinophilic granuloma; asthma, chronic bronchitis,allergic rhinitis, ARDS, chronic obstructive pulmonary disease,silicosis, pulmonary sarcoidosis, pleurisy, alveolitis, vasculitis,pneumonia, bronchiectasis, pulmonary oxygen toxicity; reperfusion injuryof the myocardium, brain, or extremities; cystic fibrosis; keloidformation, scar tissue formation; atherosclerosis; systemic lupuserythematosus, autoimmune thyroiditis, multiple sclerosis; Reynaud'ssyndrome; graft versus host disease, allograft rejection; chronicglomerulonephritis; inflammatory bowel disease, Crohn's disease,ulcerative colitis, necrotizing enterocolitis; inflammatory dermatoses,contact dermatitis, atopic dermatitis, psoriasis, urticaria, fever andmyalgias due to infection; meningitis, encephalitis, and brain or spinalcord injury due to minor trauma; Sjögren's syndrome; diseases involvingleukocyte diapedesis; alcoholic hepatitis; bacterial pneumonia;antigen-antibody complex mediated diseases; hypovolemic shock; Type Idiabetes mellitus; acute and delayed hypersensitivity; disease statesdue to leukocyte dyscrasia and metastasis; thermal injury; granulocytetransfusion associated syndromes; and cytokine-induced toxicity.
 32. Amethod of inhibiting poly(ADP-ribose) polymerase activity in a cell,comprising contacting said cell with an hPARP2 antagonist in an amounteffective for inhibiting expression or activity of the polypeptideaccording to claim 1.